CN112805377A

CN112805377A - Compounds for stabilizing amylases in liquids

Info

Publication number: CN112805377A
Application number: CN201980065422.8A
Authority: CN
Inventors: S·许弗; O·斯潘根伯格; A·加西亚马科斯; H·韦伯; K-S·蒂金; S·费舍尔
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2018-10-05
Filing date: 2019-09-24
Publication date: 2021-05-14
Also published as: US20220112479A1; BR112021006317A2; EP3861114A1; WO2020069914A1; JP2022512599A; MX2021003931A

Abstract

The present invention relates to an enzyme preparation comprising component (a): at least one compound of the general formula (I), wherein the variables of the formula (I) are as follows: r¹Selected from H and C₁‑C₁₀Alkylcarbonyl, wherein the alkyl may be linear or branched and may carry one or more hydroxyl groups; r²、R³、R⁴Independently of each other, selected from H, linear C₁‑C₈Alkyl and branched C₃‑C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆‑C₁₀Aryl and C₆‑C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁‑C₈Alkyl or branched C₃‑C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H; a component (b): at least one enzyme selected from hydrolases (EC 3), preferably at least one enzyme selected from amylases, more preferably at least one enzyme selected from alpha-amylases (EC 3.2.1.1); and/or at least one enzyme is selected from proteases, preferably from subtilisin-type proteases (EC 3.4.21.62); and optionally component (c): at least one compound selected from the group consisting of solvents, enzyme stabilizers different from component (a) and compounds stabilizing the liquid enzyme preparation itself.

Description

Compounds for stabilizing amylases in liquids

The present invention relates to an enzyme preparation, preferably a liquid enzyme preparation, comprising:

Component (a): at least one compound of the general formula (I):

wherein the variables in formula (I) are as follows:

R¹selected from H and C₁-C₁₀Alkylcarbonyl, where the alkyl radical may be linear or branched and may carry one or more hydroxyl groups,

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₅Alkyl and branched C₃-C₁₀Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H;

a component (b): at least one enzyme selected from hydrolases (EC 3), preferably at least one enzyme selected from amylases, more preferably at least one enzyme selected from alpha-amylases (EC 3.2.1.1); and/or at least one enzyme selected from proteases, preferably proteases of the subtilisin type (EC 3.4.21.62);

and optionally

A component (c): at least one compound selected from the group consisting of solvents, enzyme stabilizers different from component (a) and compounds stabilizing the liquid enzyme preparation itself.

Enzymes are typically produced commercially as liquid concentrates, which are typically derived from fermentation broths. If the enzyme is retained in an aqueous environment, it tends to lose its enzymatic activity, so it is conventional practice to convert it into the anhydrous form: the aqueous concentrate may be lyophilized or spray dried, for example, in the presence of a carrier material to form an aggregate. Solid enzyme products typically require "dissolution" prior to use. In order to stabilize the enzyme in a liquid product, enzyme inhibitors, preferably reversible enzyme inhibitors, are usually used to temporarily inhibit the enzyme activity until the enzyme inhibitor is released.

The problem to be solved by the present invention relates to providing a compound that helps to reduce the loss of enzymatic activity during storage of a liquid enzyme-containing product, even if the liquid enzyme-containing product comprises a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA. It is a further object of the present invention to provide an enzyme preparation which allows flexible formulation into a liquid detergent formulation or cleaning formulation with one type of enzyme or enzyme mixture.

This problem is solved by providing compounds of the general formula (I):

wherein the variables in formula (I) are as follows:

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₅Alkyl and branched C₃-C₁₀Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H; and

wherein the compound supports the retention of enzymatic activity of at least one enzyme selected from hydrolases (EC 3), preferably from amylases, during storage of the enzyme in a liquid product.

Enzyme names are known to those skilled in the art based on recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzyme names include: EC (enzyme commission) number, recommended name, alias (if any), catalytic activity And other factors; seehttp://www.sbcs.qmul.ac.uk/iubmb/enzyme/EC3/The last updated version of day 28, 6 months 2018.

In one aspect of the invention, there is provided an enzyme preparation comprising:

component (a): at least one enzyme stabilizer selected from compounds of formula (I):

wherein the variables in formula (I) are as follows:

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₅Alkyl and branched C₃-C₁₀Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H, and

and optionally

The enzyme preparation of the invention may be liquid at 20 ℃ and 101.3 kPa. Liquids include solutions, emulsions and dispersions, gels, and the like, as long as the liquid is fluid and pourable. In one embodiment of the invention, the liquid detergent formulations of the invention have a dynamic viscosity in the range of about 500-20,000 mPas, as determined by Brookfield, e.g.spindle 3 at 20rpm, with a Brookfield viscometer LVT-II at 25 ℃.

In one embodiment, liquid means that the enzyme preparation does not show visible precipitate formation or turbidity after storage of the liquid enzyme preparation, preferably after storage at 37 ℃ for at least 20 days.

Component (a)

More specifically, component (a) is a compound of general formula (I):

wherein the variables in formula (I) are defined as follows:

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H. Linear C₁-C₈Examples of alkyl groups are methyl, ethyl, n-propyl, n-butyl, n-pentyl, and the like. Branched C₃-C₈Examples of alkyl groups are 2-propyl, 2-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, isopentyl, and the like. C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Examples of aryl groups are phenyl, 1-naphthyl, 2-naphthyl, o-carboxyphenyl, m-carboxyphenyl, p-carboxyphenyl, o-hydroxyphenyl, p-hydroxyphenyl and the like.

In one embodiment, the compound of formula (I), (II), (III), (IVI) R in the compound¹Selected from H, acetyl and propionyl. In one embodiment, R in the compound of formula (I)¹Is H. In one embodiment, R in the compound of formula (I)¹Is acetyl. In one embodiment, R in the compound of formula (I)¹Is propionyl.

In one embodiment, R in the compound of formula (I)²Is H and R³、R⁴Independently of one another, from linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈An alkyl group.

In one embodiment, R in the compound of formula (I)²、R³、R⁴Wherein R is²、R³、R⁴Selected from the group consisting of linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈An alkyl group.

In one embodiment, R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, phenylmethyl, and o-carboxyphenyl (salicyl).

In one embodiment, R in the compound of formula (I)¹、R²And R³Is H and R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C ₂An alkyl group. In one embodiment, R in the compound of formula (I)¹And R²Is H and R³And R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂An alkyl group.

In one embodiment, formula (II) is(I) R in the compound¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄An alkyl group.

Component (a) includes salts of the compounds of formula (I). Salts include alkali metal and ammonium salts such as those of mono-and triethanolamine. Potassium and sodium salts are preferred.

In one embodiment of the present invention, the enzyme preparation, preferably the liquid enzyme preparation, comprises component (a) in an amount in the range of 0.1 to 30 wt. -%, relative to the total weight of the enzyme preparation. The enzyme preparation may comprise component (a) in an amount in the range of 0.1-15 wt. -%, 0.25-10 wt. -%, 0.5-6 wt. -% or 1-3 wt. -%, all relative to the total weight of the enzyme preparation.

In one embodiment of the present invention, compound (a) comprises at least one at least partially hydrolyzed derivative of compound (a) as an impurity. In one embodiment of the present invention, component (a) comprises as impurities the following fully hydrolyzed compound (a'):

wherein the variable R¹、R²、R³And R⁴The same as described above for component (a).

The impurities may constitute up to 50 mol%, preferably 0.1 to 20 mol%, even more preferably 1 to 10 mol% of component (a). Although impurities may originate from the synthesis of component (a) and may be removed by purification methods, it is preferred not to remove it.

Component (b)

In one aspect of the invention, at least one enzyme comprised in component (b) is part of a liquid enzyme concentrate. By "liquid enzyme concentrate" is meant herein any liquid enzyme-containing product comprising at least one enzyme. "liquid" in the context of an enzyme concentrate relates to the appearance of the material at 20 ℃ and 101.3 kPa.

The liquid enzyme concentrate may result from the dissolution of solid enzyme in a solvent. The solvent may be selected from water and organic solvents. The liquid enzyme concentrate resulting from the dissolution of the solid enzyme in the solvent may contain an amount of enzyme up to a saturation concentration.

By dissolved herein is meant that the solid compound is liquefied by contact with at least one solvent. Dissolution means that the solid compound is completely dissolved in a prescribed solvent to a saturated concentration, in which no phase separation occurs.

In one aspect of the invention, component (b) of the resulting enzyme concentrate may be free of water, meaning that an insignificant amount of water is present. By an insignificant amount of water is meant herein that the enzyme preparation comprises less than 25 wt.%, less than 20 wt.%, less than 15 wt.%, less than 10 wt.%, less than 7 wt.%, less than 5 wt.%, less than 4 wt.%, less than 3 wt.%, less than 2 wt.% of water, all relative to the total weight of the enzyme concentrate, or is free of water. In one embodiment, an enzyme concentrate that is free of water means that the enzyme concentrate does not comprise a significant amount of water, but does comprise an organic solvent in an amount of 30-80 wt.%, relative to the total weight of the enzyme concentrate.

The liquid enzyme concentrate comprising water may be referred to as "aqueous enzyme concentrate". The aqueous enzyme concentrate may be an enzyme-containing solution in which the solid enzyme product has been dissolved in water. In one embodiment, "aqueous enzyme concentrate" refers to an enzyme-containing product produced from an enzyme by fermentation.

Fermentation refers to the process of culturing a recombinant cell expressing a desired enzyme in a suitable nutrient medium, thereby allowing the recombinant host cell to grow (this process may be referred to as fermentation) and express the desired protein. At the end of the fermentation, the fermentation broth is typically collected and further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction. Depending on whether the enzyme has been secreted into the liquid fraction, the desired protein or enzyme may be recovered from the liquid fraction or the cell lysate of the fermentation broth. The recovery of the desired enzyme is carried out using methods known to the person skilled in the art. Suitable methods for recovering the protein or enzyme from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, leaching, and precipitation.

The liquid enzyme concentrate may comprise the enzyme in an amount in the range of 0.1-40 wt.%, or 0.5-30 wt.%, or 1-25 wt.%, or 3-25 wt.%, or 5-25 wt.%, all relative to the total weight of the enzyme concentrate. In one embodiment, the liquid enzyme concentrate is obtained from fermentation and is aqueous.

The aqueous enzyme concentrate resulting from the fermentation may comprise water in an amount greater than about 50 wt%, greater than about 60 wt%, greater than about 70 wt%, or greater than about 80 wt%, all relative to the total weight of the enzyme concentrate. The aqueous enzyme concentrate resulting from the fermentation may contain residual components such as salts from the fermentation medium, cell debris from the production host cell, metabolites produced by the production host cell during the fermentation process. In one embodiment, the residual components may be contained in the liquid enzyme concentrate in an amount of less than 30 wt.%, less than 20 wt.%, less than 10 wt.%, or less than 5 wt.%, all relative to the total weight of the aqueous enzyme concentrate.

At least one enzyme comprised in component (b) is selected from hydrolases (EC 3), hereinafter also referred to as enzymes (component (b)). Preferred enzymes are selected from the group consisting of enzymes acting on ester bonds (e.c.3.1), glycosylases (e.c.3.2) and peptidases (e.c. 3.4). The enzyme acting on the ester bond (E.C.3.1) is also referred to below as lipase. Glycosylases (e.c.3.2) are also referred to below as amylases, cellulases and mannanases (mannanases). Peptidases are also referred to below as proteases.

The hydrolase comprised in component (b) is identified by a polypeptide sequence (also referred to herein as an amino acid sequence). The polypeptide sequence defines a three-dimensional structure that includes the "active site" of the enzyme, which in turn determines the catalytic activity of the enzyme. The polypeptide sequence may be identified by SEQ ID NO. According to the World Intellectual Property Office (WIPO) standard st.25(1998), amino acids are indicated herein using the three-letter code in upper case letters or the corresponding single letter.

Any enzyme comprised in component (b) of the present invention (component (b)) relates to a parent enzyme and/or a variant enzyme, both having enzymatic activity. An enzyme with enzymatic activity is enzymatically active or produces an enzymatic conversion, which means that the enzyme acts on a substrate and converts these into products. The term "enzyme" herein does not include inactive variants of the enzyme.

A "parent" sequence (of a parent protein or enzyme, also referred to as a "parent enzyme") is a starting sequence for introducing changes to the sequence (e.g., by introducing one or more amino acid substitutions, insertions, deletions, or combinations thereof) to result in a "variant" of the parent sequence. The term parent enzyme (or parent sequence) includes both the wild-type enzyme (sequence) and synthetically produced sequences (enzymes) which serve as starting sequences for introducing (other) changes.

The term "enzyme variant" or "sequence variant" or "variant enzyme" relates to an enzyme that differs to some extent in its amino acid sequence from its parent enzyme. If not indicated to the contrary, a variant enzyme having "enzymatic activity" means that the variant enzyme has the same type of enzymatic activity as the corresponding parent enzyme.

The nomenclature described below is used in describing the variants of the invention:

amino acid substitutions are described as follows: the original amino acids of the parent enzyme are provided, followed by position numbering in the amino acid sequence, followed by the substituted amino acids.

Amino acid deletions are described as follows: providing the original amino acid of the parent enzyme, followed by position numbering in the amino acid sequence, followed by.

Amino acid insertions are described as follows: the original amino acids of the parent enzyme are provided, followed by position numbering in the amino acid sequence, followed by the original amino acids and additional amino acids. For example, the insertion of a lysine at position 180 immediately following glycine is denoted as "Gly 180 GlyLys" or "G180 GK".

In the case where substitution and insertion occur at the same position, this may be denoted as S99SD + S99A or simply S99 AD. In the case where an amino acid residue identical to an existing amino acid residue is inserted therein, it is clear that the nomenclature degeneracy appears. If for example glycine is inserted after glycine in the above examples this will be indicated by G180 GG.

When different changes can be introduced at a position, these different changes are separated by commas, e.g. "Arg 170Tyr, Glu" indicates that the arginine at position 170 is replaced by tyrosine or glutamic acid. Or different alterations or optional substitutions may be shown in parentheses, for example Arg170[ Tyr, Gly ] or Arg170{ Tyr, Gly }; or simply R170[ Y, G ] or R170{ Y, G }; or the full length is represented as R170Y, R170G.

Enzyme variants may be defined by their sequence identity when compared to the parent enzyme. Sequence identity is typically provided in "% sequence identity" or "% identity". To calculate sequence identity, a sequence alignment must be generated in a first step. According to the invention, a pairwise overall alignment must be generated, which means that two sequences must be aligned over their full length, which is usually generated using a mathematical method called an alignment algorithm.

According to the present invention, alignments are generated using the algorithm of Needleman and Wunsch (J.mol.biol. (1979)48, page 443-. The program "needlele" (The European Molecular Biology Open Software Suite (EMBOSS)) is preferably used for The purposes of The present invention using program default parameters (gap Open 10.0, gap extension 0.5 and matrix EBLOSUM 62).

The following calculation of% identity is applied according to the invention: percent identity-100 (identical residues/length of aligned region showing the corresponding sequence of the invention over its entire length).

Enzyme variants according to the invention may be described as amino acid sequences which are at least n% identical to the amino acid sequence of the corresponding parent enzyme, wherein "n" is an integer from 10 to 100. In one embodiment, the variant enzyme is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical when compared to the full length amino acid sequence of the parent enzyme, wherein the enzyme variant has enzymatic activity.

Enzyme variants may be defined by their sequence similarity when compared to the parent enzyme. Sequence similarity is typically provided as "% sequence similarity" or "% similarity". % sequence similarity takes into account that a defined group of amino acids share similar properties, such as their size, their hydrophobicity, their charge, or other characteristics. The exchange of an amino acid for a similar amino acid may be referred to herein as a "conservative mutation".

To determine% similarity according to the present invention, the following applies: amino acid a is similar to amino acid S; amino acid D is similar to amino acids E and N; amino acid E is similar to amino acids D, K and Q; amino acid F is similar to amino acids W and Y; amino acid H is similar to amino acids N and Y; amino acid I is similar to amino acids L, M and V; amino acid K is similar to amino acids E, Q and R; amino acid L is similar to amino acids I, M and V; amino acid M is similar to amino acids I, L and V; amino acid N is similar to amino acids D, H and S; amino acid Q with amino acids E, K and R; amino acid R is similar to amino acids K and Q; amino acid S is similar to amino acids A, N and T; amino acid T is similar to amino acid S; amino acid V is similar to amino acids I, L and M; amino acid W is similar to amino acids F and Y; amino acid Y is similar to amino acids F, H and W.

Conservative amino acid substitutions may occur over the full length of the polypeptide sequence of a functional protein, such as an enzyme. In one embodiment, such mutations do not involve a functional domain of the enzyme. In one embodiment, the conservative mutation does not involve the catalytic center of the enzyme.

To account for conservative mutations, the value of sequence similarity of two amino acid sequences can be calculated from the same alignment, which is used to calculate% identity.

The following calculation of% similarity is applied according to the invention: % similarity is ═ 100 [ (identical residues + similar residues)/length of aligned region showing the corresponding sequence of the invention over its entire length ].

Enzyme variants according to the invention may be described as amino acid sequences which are at least m% similar to the corresponding parent sequence, wherein "m" is an integer from 10 to 100. In one embodiment, the variant enzyme is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme, wherein the variant enzyme has enzymatic activity.

"enzymatic activity" refers to the catalytic effect exerted by an enzyme, usually expressed in units per milligram of enzyme (specific activity), the latter relating to the number of substrate molecules converted per minute per molecule of enzyme (molecular activity).

A variant enzyme may have enzymatic activity according to the invention when said enzyme variant shows at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the enzymatic activity of the corresponding parent enzyme.

Component (b) preferably comprises at least one hydrolase selected from the group consisting of amylases.

Amylase

The "amylases" (alpha and/or beta) of the invention include those of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably component (b) comprises at least one enzyme selected from the group consisting of alpha-amylases (EC 3.2.1.1). Chemically modified or protein engineered mutants are included.

The amylases comprised in component (b) of the present invention have an "amylolytic activity" or "amylase activity" which is related to the (intra) hydrolysis of the glucosidic bonds in the polysaccharide. Alpha-amylase activity can be determined by assays known to those skilled in the art for measuring alpha-amylase activity. Examples of assays for measuring alpha-amylase activity are:

the alpha-amylase activity can be measured by a method using Phadebas tablets as a substrate (Phadebas amylase test, supplied by Magle Life Science). The starch is hydrolyzed by the alpha-amylase to give a soluble blue fragment. The absorbance of the resulting blue solution, measured spectrophotometrically at 620nm, is a function of the alpha-amylase activity. The measured absorbance is directly proportional to the specific activity of the alpha-amylase (activity/mg of pure alpha-amylase protein) under a given set of conditions.

The alpha-amylase activity can also be determined by a method using ethylene-4-nitrophenyl-alpha-D-maltoheptaside (EPS). D-maltoheptaside is a protected oligosaccharide that can be cleaved by endoamylase. After lysis, the alpha-glucosidase enzyme included in the kit digests the substrate to release free PNP molecules, which are yellow and thus can be measured by visible spectrophotometry at 405 nm. A kit containing an EPS substrate and alpha-glucosidase was manufactured by Roche Costum Biotech (catalog No. 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity of the alpha-amylase (activity/mg enzyme) under a given set of conditions.

Amylolytic activity may be provided in units per gram of enzyme. For example, 1 unit of alpha-amylase at pH 6.9 and 20 ℃ can release 1.0mg maltose from starch within 3 minutes.

The at least one amylase comprised in component (b) may be selected from the following:

an amylase from Bacillus licheniformis (Bacillus licheniformis) as described in WO 95/10603 having SEQ ID NO: 2. Suitable variants comprising one or more substitutions at the following positions are described in WO 95/10603: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444, which have amylolytic activity. Variants are described in SEQ ID NO. 4 of WO 94/02597, WO 94/018314, WO 97/043424 and WO 99/019467.

An amylase from Bacillus stearothermophilus (B.stearothermophilus) or an amylase optionally with a C-terminal truncation in the wild type sequence as disclosed in WO 02/10355 having SEQ ID NO 6. Suitable variants of SEQ ID NO 6 include those comprising a deletion in position 181 and/or 182 and/or a substitution in position 193.

An amylase from Bacillus (Bacillus sp.)707 having SEQ ID NO:6 as disclosed in WO 99/19467. Preferred variants of SEQ NO 6 are those having substitutions, deletions or insertions in one or more of the following positions: r181, G182, H183, G184, N195, I206, E212, E216 and K269.

An amylase from Bacillus gorgeous (Bacillus halmapalus) having SEQ ID NO 2 or SEQ ID NO 7 as described in WO 96/23872, also described herein as SP-722. Preferred variants are described in WO 97/3296, WO 99/194671 and WO 2013/001078.

An amylase from Bacillus DSM 12649 as disclosed in WO 00/22103 having SEQ ID NO: 4.

An amylase from Bacillus strain TS-23 having SEQ ID NO 2 as disclosed in WO 2009/061380.

An amylase from the genus Cytophaga (Cytophaga sp.) having SEQ ID NO:1 as disclosed in WO 2013/184577.

An amylase from Bacillus megaterium DSM 90 having SEQ ID NO:1 as disclosed in WO 2010/104675.

An amylase from the genus Bacillus comprising amino acids 1-485 of SEQ ID NO 2 as described in WO 00/60060.

An amylase from Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or a variant thereof, preferably selected from the group of amylases according to SEQ ID NO 3 as described in WO 2016/092009.

An amylase having SEQ ID NO 12 as described in WO 2006/002643 or an amylase variant comprising the substitutions Y295F and M202LITV in said SEQ ID NO 12.

An amylase having SEQ ID NO. 6 as described in WO 2011/098531 or an amylase variant comprising a substitution in said SEQ ID NO. 6 at one or more positions selected from the group consisting of: 193[ G, A, S, T or M ], 195[ F, W, Y, L, I or V ], 197[ F, W, Y, L, I or V ], 198[ Q or N ], 200[ F, W, Y, L, I or V ], 203[ F, W, Y, L, I or V ], 206[ F, W, Y, N, L, I, V, H, Q, D or E ], 210[ F, W, Y, L, I or V ], 212[ F, W, Y, L, I or V ], 213[ G, A, S, T or M ] and 243[ F, W, Y, L, I or V ].

An amylase as described in WO 2013/001078 having SEQ ID NO:1 or an amylase variant comprising alterations in two or more (several) positions in said SEQ ID NO:1 corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477.

An amylase having SEQ ID No. 2 as described in WO 2013/001087 or an amylase variant comprising a deletion in said SEQ ID No. 2 of position 181+182 or 182+183 or 183+184, optionally comprising one or two or more modifications in any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 in said SEQ ID No. 2.

An amylase which is a hybrid alpha-amylase, e.g. from the above-mentioned amylase as in WO 2006/066594;

a hybrid amylase according to WO 2014/183920, wherein the A and B domains have at least 90% identity to SEQ ID NO. 2 of WO 2014/183920 and the C domain has at least 90% identity to SEQ ID NO. 6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO 23 of WO 2014/183920 and has amylolytic activity;

a hybrid amylase according to WO 2014/183921, wherein the A and B domains have at least 75% identity to SEQ ID NO 2, SEQ ID NO 15, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 32 and SEQ ID NO 39 and the C domain has at least 90% identity to SEQ ID NO 6 of WO 2014/183921 as disclosed in WO 2014/183921, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO 30 as disclosed in WO 2014/183921 and has amylolytic activity.

Suitable amylases comprised in component (b) include amylase variants of the amylases disclosed herein having an amylase activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed above.

Suitable amylases comprised in component (b) include amylase variants of the amylases disclosed herein having an amylase activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme.

In one embodiment, the at least one amylase is selected from commercially available amylases, including but not limited to those sold under the trademark Duramyl ^TM，Termamyl^TM，Fungamyl^TM，Stainzyme^TM，Stainzyme Plus^TM，Natalase^TMLiquozyme X and BAN^TM，Amplify^TM，Amplify Prime^TM(from Novozymes A/S) and Rapidase^TM，Purastar^TM，Powerase^TM，Effecten^TM(M100, from DuPont), Preferenz^TM(S1000, S110 and F1000; from DuPont), PrimaGreen^TM(ALL；DuPont)，Optisize^TM(DuPont).

According to the present invention, component (b) may comprise a combination of at least two amylases as disclosed above, wherein the combination comprises one or more amylases selected from the group consisting of:

an amylase from Bacillus 707 or a variant thereof having amylolytic activity, preferably selected from the group consisting of an amylase having SEQ ID NO 6 as disclosed in WO 99/19467 and a variant thereof having amylolytic activity;

an amylase selected from those comprising amino acids 1-485 of SEQ ID NO 2 as described in WO 00/60060, those having SEQ ID NO 12 as described in WO 2006/002643 and variants thereof having amylolytic activity;

an amylase from Bacillus caldus crescens or a variant thereof having amylolytic activity, preferably selected from the group consisting of an amylase having SEQ ID NO:1 and 2 as disclosed in WO 2013/001078, an amylase having SEQ ID NO:6 as described in WO 2011/098531 and a variant thereof having amylolytic activity;

an amylase from Bacillus amyloliquefaciens or a variant thereof having amylolytic activity, preferably selected from the group consisting of the amylases according to SEQ ID NO. 3 of WO 2016/092009;

a hybrid amylase according to WO 2014/183921, wherein the A and B domains have at least 75% identity to SEQ ID NO 2, SEQ ID NO 15, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 32 and SEQ ID NO 39 and the C domain has at least 90% identity to SEQ ID NO 6 of WO 2014/183921 as disclosed in WO 2014/183921, wherein the hybrid amylase has amylolytic activity;

preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO 30 as disclosed in WO 2014/183921 and has amylolytic activity.

In one embodiment, component (b) comprises a combination of at least one amylase, preferably selected from the following, and at least one other enzyme, preferably selected from the group consisting of proteases, lipases, cellulases and mannanases:

Protease enzyme

The at least one enzyme comprised in component (b) may be selected from proteases, preferably from serine endopeptidases (EC 3.4.21), most preferably from subtilisin-type proteases (EC 3.4.21.62).

Proteases are members of the EC 3.4 class. Proteases include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl and tripeptidyl peptidases (EC 3.4.14), peptidyl dipeptidases (EC 3.4.15), serine type carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine type carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metalloendopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25) or endopeptidases of unknown catalytic mechanism (EC 3.4.99).

In one embodiment, component (b) comprises at least one protease selected from the group consisting of serine proteases (EC 3.4.21). Serine proteases or serine peptidases are characterized by a serine at the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. Serine proteases in the context of the present invention may be selected from chymotrypsin (e.g. EC 3.4.21.1), elastase (e.g. EC 3.4.21.36), elastase (e.g. EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g. EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g. EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118 or EC 3.4.21.119), plasmin (e.g. EC 3.4.21.7), trypsin (e.g. EC 3.4.21.4), thrombin (e.g. EC 3.4.21.5) and subtilisin. Subtilisins are also known as subtilisins, for example EC 3.4.21.62, the latter also being referred to below as "subtilisins".

The serine protease subgroup provisionally referred to as subtilases has been proposed by Siezen et al (1991), Protein Eng.4:719-737 and Siezen et al (1997), Protein Science 6: 501-523. Subtilases include the subtilisin family, the thermotolerant protease family, the proteinase K family, the lantibiotic peptidase family, the Kexin family, and the hyperthermophilic protease family.

The subtilase subgroup is subtilisin, which is derived from the MEROPS database: (http:// merops.sanger.ac.uk) A serine protease of family S8 as defined. Peptidase family S8 includes the serine endopeptidase subtilisin and its homologs. In subfamily S8A, the active site residues are usually found in the motifs Asp-Thr/Ser-Gly (similar to those in the aspartate endopeptidase family in heterogenous group AA), His-Gly-Thr-His, and Gly-Thr-Ser-Met-Ala-Xaa-Pro.

The subtilisin-related class of serine proteases share a common amino acid sequence that defines the catalytic triad, which distinguishes them from the chymotrypsin-related class of serine proteases. Both subtilisin and chymotrypsin related serine proteases have catalytic triads including aspartic acid, histidine and serine.

Examples include subtilisin as described in WO 89/06276 and EP 0283075, WO 89/06279, WO 89/09830, WO 89/09819, WO 91/06637 and WO 91/02792.

Proteases are active proteins that exert a "protease activity" or "proteolytic activity". Proteolytic activity is related to the rate at which a protein is degraded by a protease or proteolytic enzyme over a defined period of time.

Methods for assaying proteolytic activity are well known in the literature (see, e.g., Gupta et al (2002), appl. Microbiol. Biotechnol.60: 381-395). The proteolytic activity can be increased by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(Suc-AAPF-pNA, abbreviated AAPF; see, for example, DelMar et al (1979), Analytical Biochem 99, 316-. pNA is separated from the substrate molecule by proteolytic cleavage, resulting in the release of yellow free pNA, which can be measured by OD₄₀₅And quantized.

Proteolytic activity may be provided in units per gram of enzyme. For example, 1U protease may correspond to an amount of protease (casein as substrate) that releases 1. mu. mol of forskolin positive amino acids and peptides (as tyrosine) per minute at pH 8.0 and 37 ℃.

The protease of the subtilisin type (EC 3.4.21.62) may be a microbial-derived bacterial protease selected from the group consisting of: bacillus (Bacillus), Clostridium (Clostridium), Enterococcus (Enterococcus), Geobacillus (Geobacillus), Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), marine Bacillus (Oceanobacillus), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus) or Streptomyces (Streptomyces) proteases, or gram-negative bacterial polypeptides such as Campylobacter (Campylobacter), escherichia coli (e.coli), Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Helicobacter (Helicobacter), corynebacterium (corynebacterium), Neisseria (Neisseria), Pseudomonas (yodomonas), Salmonella (Salmonella) and Ureaplasma (Ureaplasma).

In one aspect of the invention, the at least one protease is selected from the group consisting of Bacillus alcalophilus (Bacillus alcalophilus), Bacillus amyloliquefaciens, Bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus coagulosus), Bacillus firmus (Bacillus firmus), Bacillus gibsonii (Bacillus gibsonii), Bacillus lautus (Bacillus lautus), Bacillus lentus (Bacillus lentus), Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus (Bacillus pumilus), Bacillus sphaericus (Bacillus sphaericus), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus subtilis), and Bacillus thuringiensis (Bacillus thuringiensis) proteases.

In one embodiment of the present invention, component (b) comprises at least one protease selected from the group consisting of: subtilisin from Bacillus amyloliquefaciens BPN' (described by Vasantha et al (1984) J. bacteriol., 159, p. 811-819 and JA Wells et al (1983) in Nucleic Acids Research, Vol. 11, p. 7911-7925); subtilisin from Bacillus licheniformis (subtilisin Carlsberg; disclosed in EL Smith et al (1968), J.biol Chem, Vol.243, pp.2184-2191 and Jacobs et al (1985), Nucl.acids Res, Vol.13, pp.8913-8926); subtilisin PB92 (the original sequence of alkaline protease PB92 is described in EP 283075a 2); subtilisin 147 and/or 309 (respectively: subtilisin 147 and/or 309) disclosed in WO 89/06279

) (ii) a A subtilisin from Bacillus lentus as disclosed in WO 91/02792, such as subtilisin from Bacillus lentus DSM 5483 or a variant of Bacillus lentus DSM 5483 described in WO 95/23221; subtilisin from Bacillus alkalophilus (DSM 11233) disclosed in DE 10064983; subtilisin from bacillus gibsonii (DSM 14391) disclosed in WO 2003/054184; subtilisin from Bacillus (DSM 14390) disclosed in WO 2003/056017; subtilisin from Bacillus (DSM 14392) disclosed in WO 2003/055974; subtilisin from bacillus gibsonii (DSM 14393) disclosed in WO 2003/054184; subtilisin having SEQ ID NO 4 as described in WO 2005/063974; subtilisin having SEQ ID NO 4 as described in WO 2005/103244; subtilisin having SEQ ID NO 7 as described in WO 2005/103244; and subtilisin with SEQ ID NO 2 as described in application DE 102005028295.4.

In one embodiment, component (b) comprises at least subtilisin 309 (which may be referred to herein as Savinase) disclosed in table I of WO 89/06279 as sequence a) or a variant thereof which is at least 80% identical thereto and has proteolytic activity.

Examples of proteases useful according to the invention include variants described in WO 92/19729, WO 95/23221, WO 96/34946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO 02/088340, WO 03/006602, WO 2004/03186, WO 2004/041979, WO 2007/006305, WO 2011/036263, WO 2011/036264 and WO 2011/072099. Suitable examples include in particular variants of subtilisin protease derived from SEQ ID NO:22 as described in EP 1921147 (being the sequence of the mature alkaline protease from B.lentus DSM 5483) with amino acid substitutions in one or more of the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (numbered according to BPN'), which have proteolytic activity. In one embodiment, the protease is not mutated at positions Asp32, His64 and Ser221 (numbering according to BPN').

Component (b) may comprise protease variants having proteolytic activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed above.

Component (b) may comprise protease variants having proteolytic activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme.

In one embodiment, at least one protease comprised in component (b) has SEQ ID NO 22 as described in EP 1921147, or is a protease which is at least 80% identical thereto and has proteolytic activity. In one embodiment, the protease is characterized by the amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (a) or glycine (G), or serine (S) at position 101 (numbering according to BPN') and has proteolytic activity. In one embodiment, the protease comprises one or more further substitutions: (a) threonine (3T) at position 3, (b) isoleucine (4I) at position 4, (c) alanine, threonine or arginine (63A, 63T or 63R) at position 63, (D) aspartic acid or glutamic acid (156D or 156E) at position 156, (E) proline (194P) at position 194, (f) methionine (199M) at position 199, (G) isoleucine (205I) at position 205, (h) aspartic acid, glutamic acid or glycine (217D, 217E or 217G) at position 217, and (I) combinations of two or more amino acids according to (a) - (h). At least one protease may be at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by comprising one amino acid (according to (a) - (h)) or a combination according to (i) together with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (numbering according to BPN') and having proteolytic activity. In one embodiment, the protease is characterized by comprising a mutation (numbering according to BPN') R101E or S3T + V4I + V205I, or R101E and S3T, V4I and V205I, or S3T + V4I + V199M + V205I + L217D and having proteolytic activity.

In one embodiment, the protease according to SEQ ID No. 22 as described in EP 1921147 is characterized by comprising the mutation (numbering according to BPN') S3T + V4I + S9R + a15T + V68A + D99S + R101S + a103S + I104V + N218D and having proteolytic activity.

In one embodiment, the at least one protease is selected from commercially available proteases, including but not limited to those sold under the trade designation

Duralase^TM，Durazym^TM，

Ultra，

Ultra，

Ultra，

Ultra，

And

(Novozymes A/S) sold under the brand name

Prime，Purafect

Purafect

Purafect

And

(Danisco/DuPont)，Axapem^TM(Gist-Brocases N.V.) Bacillus lentus alkaline protease (BLAP; sequence shown in FIG. 29 of US 5,352,604) and variants thereof and KAP (Bacillus alkalophilus subtilisin) from Kao Corp.

According to the invention, component (b) may comprise a combination of at least two proteases, preferably a protease selected from the group of serine endopeptidases (EC 3.4.21), more preferably from the group of subtilisin-type proteases (EC 3.4.21.62) -all as disclosed above.

In one embodiment, component (b) comprises at least one protease selected from the group consisting of the protease according to SEQ ID NO:22 as described in EP 1921147 or a variant thereof having proteolytic activity as disclosed above.

In one embodiment, component (b) comprises at least one protease selected from subtilisin 309 disclosed in WO 89/06279 table I a) or a proteolytically active variant thereof as disclosed above.

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of the following and at least one protease as disclosed above, preferably a protease selected from the group consisting of serine endopeptidases (EC 3.4.21), more preferably a protease selected from the group consisting of subtilisin type proteases (EC 3.4.21.62):

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of, at least one protease as disclosed above, preferably a protease selected from the group consisting of serine endopeptidases (EC 3.4.21), more preferably a protease selected from the group consisting of subtilisin type proteases (EC 3.4.21.62) and at least one further enzyme preferably selected from the group consisting of lipases, cellulases and mannanases:

Lipase enzyme

The at least one enzyme comprised in component (b) may be selected from lipases. "Lipase", "lipolytic enzyme", "lipid esterase" all relate to enzymes of EC class 3.1.1 ("carboxylic ester hydrolases"). Lipase refers to an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; the enzyme having cutinase activity may be referred to herein as cutinase), sterol esterase activity (EC 3.1.1.13), and/or wax ester hydrolase activity (EC 3.1.1.50).

Methods for determining lipolytic activity are well known in the literature (see e.g.Gupta et al (2003), Biotechnol. appl. biochem.37, pages 63-71). For example, lipase activity can be measured by hydrolysis of the ester bond in the substrate p-nitrophenylpalmitate (pNP palmitate, C:16) and release of pNP, which is yellow and can be detected at 405 nm.

"lipolytic activity" refers to the catalytic effect produced by a lipase, which may be provided in Lipolytic Units (LU). For example, 1LU may correspond to the amount of lipase producing 1. mu. mol titratable fatty acids per minute at constant pH under the following conditions: the temperature is 30 ℃; pH 9.0; the substrate may be 3.3 weightAmount% olive oil and 3.3% gum arabic at 13mmol/l Ca²⁺And an emulsion in 5mmol/l Tris buffer in the presence of 20mmol/l NaCl.

Lipases which may be contained in component (b) include those of bacterial or fungal origin. In one aspect of the invention, suitable lipases (component (b)) are selected from the following: lipases from the genus Humicola (Humicola), synonymously thermophila (Thermomyces), for example from Humicola lanuginosa (h.lanuginosa) as described in EP 258068, EP 305216, WO 92/05249 and WO 2009/109500 or from Humicola insolens (h.insolens) as described in WO 96/13580; lipases derived from Rhizomucor miehei (Rhizomucor miehei) as described in WO 92/05249; lipases from strains of the genus Pseudomonas (some of these are now renamed Burkholderia), for example from Pseudomonas alcaligenes (p.alcaligenes) or Pseudomonas pseudoalcaligenes (p.pseudoalcaligenes) (EP 218272, WO 94/25578, WO 95/30744, WO 95/35381, WO 96/00292), Pseudomonas cepacia (p.cepacia) (EP 331376), Pseudomonas stutzeri (GB 1372034), Pseudomonas fluorescens (p.fluoroscecens), Pseudomonas strain SD705(WO 95/06720 and WO 96/27002), Pseudomonas wisconsisitins (p.wisconsinensis) (WO 96/12012), Pseudomonas mendocina (Pseudomonas mendocina) (WO 95/14783), Pseudomonas glumae (p.glumae) (WO 95/35381, WO 96/00292); lipases from Streptomyces griseus (WO 2011/150157) and Streptomyces pristinaespiralis (WO 2012/137147), GDSL-type Streptomyces lipases (WO 2010/065455); lipases from Thermobifida fusca as disclosed in WO 2011/084412; lipases from Geobacillus stearothermophilus (Geobacillus stearothermophilus) as disclosed in WO 2011/084417; for example, Bacillus lipases as disclosed in WO 00/60063, such as those from Bacillus subtilis (B.subtilis), Bacillus stearothermophilus (JP S64-074992) or Bacillus pumilus (B.pumilus) (WO 91/16422) as disclosed in Dartois et al (1992), Biochemica et Biophysica Acta, 1131, 253-360 or WO 2011/084599; lipases from Candida antarctica (Candida antarctica) as disclosed in WO 94/01541; cutinases from pseudomonas mendocina (US 5389536, WO 88/09367); cutinases from Magnaporthe grisea (WO 2010/107560); cutinases from fusarium Sun (Fusarium solani pisi) as disclosed in WO 90/09446, WO 00/34450 and WO 01/92502; and cutinases from Humicola lanuginosa (Humicola lanuginosa) as disclosed in WO 00/34450 and WO 01/92502.

Suitable lipases also include those known as acyltransferases or perhydrolases, for example acyltransferases homologous to candida antarctica lipase a (WO 2010/111143), acyltransferases from Mycobacterium smegmatis (Mycobacterium smegmatis) (WO 2005/056782), perhydrolases from the CE7 family (WO 2009/67279) and variants of Mycobacterium smegmatis (m. smegmatis) perhydrolases, especially the S54V variant (WO 2010/100028).

Component (b) may comprise a lipase variant having lipolytic activity of the above-mentioned lipase. Suitable lipase variants of this type are, for example, those which have been cultivated by the processes disclosed in WO 95/22615, WO 97/04079, WO 97/07202, WO 00/60063, WO 2007/087508, EP 407225 and EP 260105.

Component (b) may comprise lipase variants having lipolytic activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed above.

Component (b) may comprise lipase variants having lipolytic activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme as disclosed above.

In one embodiment, component (b) comprises at least one lipase selected from the group consisting of fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from Thermomyces lanuginose (Thermomyces lanuginose) lipase. In one embodiment, the Thermomyces lanuginosus lipase is selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity. The triacylglycerol lipase according to amino acids 1 to 269 of SEQ ID NO. 2 of US 5869438 may be referred to herein as Lipolase.

Thermomyces lanuginosus lipase may be selected from variants having lipolytic activity which are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

Thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising only conservative mutations, but they do not relate to the domain of amino acids 1-269 of SEQ ID NO. 2 of US 5869438. The lipase variants with lipolytic activity of this embodiment may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO. 2 of US 5869438.

Thermomyces lanuginosus lipase may be at least 80% identical to SEQ ID NO 2 of US 5869438, which is characterized by the amino acids T231R and N233R. The thermomyces lanuginosus lipase may further comprise one or more of the following amino acid exchanges: Q4V, V60S, a150G, L227G, P256K.

In one embodiment, at least one lipase is selected from commercially available lipases including, but not limited to, Lipolase under the trademark LIPOLASE^TM，Lipex^TM，Lipolex^TMAnd Lipoclean^TM(Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (Gist-Brocades/now DSM).

According to the present invention, component (b) may comprise a combination of at least two lipases, preferably a lipase selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity as disclosed above.

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of the following and at least one lipase as disclosed above, preferably a lipase selected from the group consisting of the triacylglycerol lipases according to amino acids 1-269 of SEQ ID NO:2 of US 5869438 and variants thereof having lipolytic activity:

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of at least one lipase as disclosed above, preferably a lipase selected from the group consisting of the triacylglycerol lipases according to amino acids 1 to 269 of SEQ ID No. 2 of US 5869438 and variants thereof having lipolytic activity, at least one protease as disclosed above, preferably a protease selected from the group consisting of serine endopeptidases (EC 3.4.21), more preferably a protease selected from the group consisting of subtilisin-type proteases (EC 3.4.21.62) and at least one further enzyme preferably selected from the group consisting of cellulases and mannanases:

Cellulase enzymes

The at least one enzyme comprised in component (b) may be selected from cellulases. The at least one cellulase may be selected from the group consisting of cellobiohydrolases (1, 4-P-D-glucan cellobiohydrolases, EC 3.2.1.91), endo-ss-1, 4-glucanases (endo-1, 4-P-D-glucan 4-glucanohydrolases, EC 3.2.1.4) and ss-glucosidases (EC 3.2.1.21). Preferably component (b) comprises at least one cellulase of glycosyl hydrolase family 7 (GH7, pfam00840), preferably a cellulase selected from endoglucanases (EC 3.2.1.4).

"cellulase", "cellulase" or "cellulolytic enzyme" (component (b)) are enzymes involved in the hydrolysis of cellulose. Assays for measuring "cellulase activity" or "cellulolytic activity" are known to those skilled in the art. For example, cellulolytic activity may be determined by the fact that carboxymethylcellulose is hydrolyzed by cellulase enzymes to reducing carbohydrates, the reducing capacity of the latter being determined by means of the ferricyanide reaction colorimetrically according to Hoffman, w.s., j.biol.chem.120, 51 (1937).

Cellulolytic activity may be provided in units per gram of enzyme. For example, 1 unit at pH 5.0 and 37 ℃ can release 1.0. mu. mol glucose from cellulose within 1 hour (2 hours incubation time).

Cellulases of the invention include those of bacterial or fungal origin. In one embodiment, the at least one cellulase is selected from cellulases comprising a cellulose binding domain. In one embodiment, the at least one cellulase is selected from cellulases comprising only the catalytic domain, which means that the cellulase does not have a cellulose binding domain.

In one embodiment, at least one cellulase comprised in component (b) is selected from commercially available cellulases including, but not limited to, Celluzyme^TM，Endolase^TM，Carezyme^TM，Cellusoft^TM，Renozyme^TM，Celluclean^TM(from Novozymes A/S), Ecostone^TM，Biotouch^TM，Econase^TM，Ecopulp^TM(from AB Enzymes Finland), Clazinase^TM，Puradax HA^TMGenencor detergent cellulase L, IndiAge^TMNeutra (from Genencor International Inc./DuPont), Revitalenz^TM(2000 from DuPont), Primafast^TM(DuPont) and KAC-500^TM(from Kao Corporation).

According to the invention, component (b) may comprise a combination of at least two cellulases, preferably cellulases selected from the group of endoglucanases (EC 3.2.1.4) as disclosed above.

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of:

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the following, at least one cellulase of the GH7 family, preferably a cellulase selected from the endoglucanases as disclosed above (EC 3.2.1.4), and one or more other enzymes:

A hybrid amylase according to WO 2014/183921, wherein the A and B domains have at least 75% identity to SEQ ID NO 2, SEQ ID NO 15, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 32 and SEQ ID NO 39 and the C domain has at least 90% identity to SEQ ID NO 6 of WO 2014/183921 as disclosed in WO 2014/183921, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO 30 as disclosed in WO 2014/183921 and has amylolytic activity;

said further enzyme is preferably selected from:

a lipase as disclosed above, preferably a lipase selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO. 2 according to US 5869438 and variants thereof having lipolytic activity;

a protease as disclosed above, preferably selected from serine endopeptidases (EC 3.4.21), more preferably from subtilisin-type proteases (EC 3.4.21.62); and

mannanase.

Mannose degrading enzyme

The at least one enzyme comprised in component (b) may be selected from mannan-degrading enzymes. The at least one mannose-degrading enzyme may be selected from β -mannosidase (EC 3.2.1.25), endo-1, 4- β -mannosidase (EC 3.2.1.78) and 1,4- β -mannosidase (EC 3.2.1.100). Preferably, the at least one mannose degrading enzyme is selected from the group consisting of endo-1, 4-beta-mannosidase (EC 3.2.1.78), a group of enzymes that may be referred to herein as endo-beta-1, 4-D-mannanase, beta-mannanase or mannanase.

Polypeptides having mannanase activity the mannanase activity may be tested according to standard test procedures known in the art, such as applying the test solution to a test solution containing 0.2% AZCL galactomannan (carob flour), namely a protein prepared by the company Megazyme (internet address of Megazyme:http://www.megazyme.com/Purchase/index.html) 4mm diameter wells punched out of agar plates used for analysis of endo-1, 4-beta-D-mannanase substrates, obtained under catalog number I-AZGMA.

Component (b) may comprise at least one mannanase selected from the alkaline mannanases of families 5 or 26. The term "alkaline mannanase" is intended to include mannanases having an enzymatic activity of at least 40% of their maximal activity at a given pH in the range of 7-12, preferably 7.5-10.5.

At least one mannanase contained in component (b) may be selected from the group consisting of those derived from Bacillus as described in JP-0304706[ beta-mannanase from Bacillus ], JP-63056289[ thermostable alkaline beta-mannanase ], JP-63036774[ Bacillus microorganism FERM P-8856 producing beta-mannanase and beta-mannosidase at alkaline pH ], JP-08051975[ alkaline beta-mannanase from Bacillus alkalophilus AM-001 ], WO 97/11164[ mannanase from Bacillus amyloliquefaciens ], WO 91/18974[ mannanase activity at extreme pH and temperature ], WO 97/11164[ mannanase from Bacillus amyloliquefaciens ], WO 2014100018[ endo- (3-mannanase 1(Bleman 1; see U.S.5,476,775) ] cloned from Bacillus circulans or Bacillus lentus strain CMG1240 Mannanase from a genus organism. Suitable mannanases are described in WO 99/064619.

The at least one mannanase enzyme comprised in component (b) may be selected from mannanases derived from Trichoderma (Trichoderma) organisms as disclosed in WO 93/24622.

Component (b) may comprise mannanase variants having mannanase activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the corresponding parent enzyme as disclosed above.

Component (b) may comprise mannanase variants having mannanase activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the corresponding parent enzyme as disclosed above.

Component (b) may comprise a commercially available mannanase such as

(Novozymes AIS)。

In one embodiment, component (b) comprises a combination of at least one alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of an alpha-amylase as disclosed below, at least one alkaline mannanase, preferably an alkaline mannanase selected from the group consisting of an endo-1, 4-beta-mannosidase as disclosed above (EC 3.2.1.78) and one or more other enzymes:

the further enzyme is preferably selected from:

cellulases as disclosed above, preferably cellulases of the GH7 family, more preferably cellulases selected from the group consisting of endoglucanases (EC 3.2.1.4) as disclosed above;

a lipase as disclosed above, preferably a lipase selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO. 2 according to US 5869438 and variants thereof having lipolytic activity; and. a protease as disclosed above, preferably selected from serine endopeptidases (EC 3.4.21), more preferably from subtilisin-type proteases (EC 3.4.21.62).

Component (c)

In one embodiment, the liquid enzyme formulation of the invention comprises component (c) comprising at least one compound selected from the group consisting of solvents, enzyme stabilizers different from component (a), and compounds stabilizing the liquid enzyme formulation itself.

An enzyme stabilizer different from component (a):

the liquid enzyme formulation of the present invention may comprise at least one enzyme stabilizer different from component (a). The enzyme stabilizer (component (c)) may be selected from boron containing compounds, polyols, peptide aldehydes, other stabilizers and mixtures thereof.

A boron-containing compound:

the boron-containing compound (component (c)) may be selected from boric acid or derivatives thereof and organic boric acids or derivatives thereof such as aryl boric acids or derivatives thereof, salts thereof and mixtures thereof. Boric acid may be referred to herein as orthoboric acid.

In one embodiment, the boron containing compound (component (c)) is selected from arylboronic acids and derivatives thereof. In one embodiment, the boron-containing compound is selected from the group consisting of phenylboronic acid (BBA), also known as phenylboronic acid (PBA), derivatives thereof, and mixtures thereof. In one embodiment, the phenyl boronic acid derivative is selected from the group consisting of derivatives of formula (IIIa) and formula (IIIb):

wherein

R1 is selected from hydrogen, hydroxy, unsubstituted or substituted C₁-C₆Alkyl and unsubstituted or substituted C₁-C₆An alkenyl group; in a preferred embodiment, R is selected from the group consisting of hydroxy and unsubstituted C₁An alkyl group;

r2 is selected from hydrogen, hydroxy, unsubstituted or substituted C₁-C₆Alkyl and unsubstituted or substituted C₁-C₆An alkenyl group; in a preferred embodiment, R is selected from H, hydroxy and substituted C ₁An alkyl group.

In one embodiment, the phenyl boronic acid derivative (component (c)) is selected from the group consisting of 4-formylphenyl boronic acid (4-FPBA), 4-carboxyphenyl boronic acid (4-CPBA), 4- (hydroxymethyl) phenyl boronic acid (4-HMPBA) and p-tolyl boronic acid (p-TBA).

Other suitable derivatives (component (c)) include 2-thienylboronic acid, 3-thienylboronic acid, (2-acetamidophenyl) boronic acid, 2-benzofuranylboronic acid, 1-naphthylboronic acid, 2-FPBA, 3-FBPA, 1-thianthrenylboronic acid, 4-dibenzofuranboronic acid, 5-methyl-2-thienylboronic acid, 1-benzothiophene-2-boronic acid, 2-furanylboronic acid, 3-furanylboronic acid, 4-biphenyldiboronic acid, 6-hydroxy-2-naphthaleneboronic acid, 4- (methylthio) phenylboronic acid, 4- (trimethylsilyl) phenylboronic acid, 3-bromothiopheneboronic acid, 4-methylthiothiopheneboronic acid, 2-naphthylboronic acid, 5-bromothiopheneboronic acid, 5-chlorothienylboronic acid, dimethylthiopheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiopheneboronic acid, p-methylphenylethylboronic acid, 2-thianthrylboronic acid, dibenzothiopheneboronic acid, 9-anthraceneboronic acid, 3, 5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-fluorophenylboronic acid, octylboronic acid, 1,3, 5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3, 5-bis (trifluoromethyl) phenylboronic acid, 2, 4-dichlorophenylboronic acid, 4-methoxyphenylboronic acid and mixtures thereof.

Polyol:

the polyol (component (c)) may be selected from polyols containing 2 to 6 hydroxyl groups. Suitable examples include ethylene glycol, propylene glycol, 1, 2-propanediol, 1, 2-butanediol, ethylene glycol, hexanediol, glycerol, sorbitol, mannitol, erythritol, glucose, fructose, lactose, and tetrahydrofurandiol.

Peptide aldehyde:

the peptide aldehyde (component (c)) may be selected from di-, tri-or tetrapeptide aldehydes and aldehyde analogs (in the form of B1-BO-R, wherein R is H, CH₃、CX₃、CHX₂Or CH₂X (X ═ halogen), BO is a single amino acid residue (in one embodiment, with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (in one embodiment, 1,2 or 3), optionally containing an N-terminal protecting group, or as described in WO 09/118375 and WO 98/13459, or is a protein type protease inhibitor such as RASI, BASI, WASI (bifunctional alpha-amylase/subtilisin inhibitors of rice, barley and wheat) or CI₂Or SSI.

Other stabilizers:

the other stabilizer (component (c)) may be selected from salts such as NaCl or KCl, and alkali metal salts of lactic acid and formic acid.

Other stabilizers (component (c)) may be selected from water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions, which provide such ions to the enzyme in the finished composition, as well as other metal ions (e.g. barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadyl (IV)).

Compounds stabilizing the liquid enzyme preparation itself

By a compound that stabilizes the liquid enzyme formulation itself is meant any compound in an amount effective to ensure storage stability, except for the enzyme stabilizer required to produce storage stability of the liquid formulation.

Storage stability in the context of liquid formulations generally includes both product appearance and dose uniformity to those skilled in the art.

Product appearance is affected by the pH of the product and the presence of compounds such as preservatives, antioxidants, viscosity modifiers, emulsifiers, and the like.

Dose uniformity is generally related to the uniformity of the product.

The enzyme preparation of the invention may be alkaline or have a neutral or slightly acidic pH, for example 6-14, 6.5-13, 8-10.5 or 8.5-9.0.

The liquid enzyme formulation of the invention may comprise at least one preservative. The preservative is added in an amount effective to prevent microbial contamination of the liquid enzyme preparation, preferably the aqueous enzyme preparation.

Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids, and formaldehyde-releasing agents. Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chloride, polyhexamethylene biguanide (PHMB), didecyldimethylammonium chloride (DDAC), and N- (3-aminopropyl) -N-dodecyl-1, 3-propanediamine (diamine). Non-limiting examples of suitable isothiazolinones include 1, 2-benzisothiazolin-3-one (BIT), 2-methyl-2H-isothiazolin-3-one (MIT), 5-chloro-2-methyl-2H-isothiazolin-3-one (CIT), 2-octyl-2H-isothiazolin-3-One (OIT), and 2-butylbenzo [ d [ ]Isothiazoles-3-ketone (BBIT). Non-limiting examples of suitable organic acids include benzoic acid, sorbic acid, L- (+) -lactic acid, formic acid, and salicylic acid. Non-limiting examples of suitable formaldehyde-releasing agents include N, N '-methylenedimorpholine (MBM), 2' - (hexahydro-1, 3, 5-triazine-1, 3, 5-triyl) triethanol (HHT), (ethylenedioxy) dimethanol, alpha '-trimethyl-1, 3, 5-triazine-1, 3,5(2H,4H,6H) -triethanol (HPT), 3' -methylenebis [ 5-methyl- ] -5

Oxazolidines](MBO) and cis-1- (3-chloroallyl) -3,5, 7-triaza-1-nitrogen

Adamantane Chloride (CTAC).

Other useful preservatives include iodopropynyl butylcarbamate (IPBC), halogen-releasing compounds such as dichlorodimethyl hydantoin (DCDMH), bromochlorodimethyl hydantoin (BCDMH), and dibromodimethyl hydantoin (DBDMH); bromonitro compounds such as Bronopol (Bronopol) (2-bromo-2-nitro-1, 3-propanediol), 2-dibromo-2-cyanoacetamide (DBNPA); aldehydes such as glutaraldehyde; phenoxyethanol; biphenyl-2-ol; and zinc or sodium pyrithione.

Solvent(s)

In one embodiment, the enzyme preparation of the invention is aqueous, comprising water in an amount in the range of 5-95 wt.%, 5-30 wt.%, 5-25 wt.% or 20-70 wt.%, all relative to the total weight of the enzyme preparation.

In one embodiment, the enzyme preparation of the invention comprises at least one organic solvent selected from the group consisting of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, ethylene glycol, propylene glycol, 1, 3-propanediol, butanediol, diethylene glycol, propyl diethylene glycol, butyl diethylene glycol, hexanediol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether and phenoxyethanol, preferably ethanol, isopropanol or propylene glycol. Further, the enzyme preparation of the present invention may comprise at least one organic solvent selected from compounds such as 2-butoxyethanol, isopropanol and d-limonene.

The enzyme preparation may comprise an organic solvent in an amount in the range of 0-20 wt% relative to the total weight of the enzyme preparation. In one embodiment, the enzyme preparation comprises water in an amount in the range of 5-15 wt% and does not comprise a significant amount of organic solvent, e.g. 1 wt% or less, all relative to the total weight of the enzyme preparation.

In one embodiment, the enzyme preparation of the invention comprises at least:

wherein the variables in formula (I) are as follows:

a component (b): at least one enzyme selected from hydrolases (EC 3), preferably at least one enzyme selected from amylases, more preferably at least one enzyme selected from alpha-amylases (EC 3.2.1.1); and/or at least one enzyme selected from proteases, preferably from subtilisin-type proteases (EC 3.4.21.62); and

a component (c): at least one enzyme stabilizer different from component (a) as disclosed above, preferably selected from polyols, peptide aldehydes and other stabilizers as disclosed above;

wherein the at least one amylase is selected from the group consisting of:

And wherein the at least one protease is selected from subtilisin-type proteases (EC 3.4.21.62), preferably from:

22 or a variant thereof having proteolytic activity according to SEQ ID No. as described in EP 1921147; and

a protease selected from subtilisin 309 as disclosed in WO 89/06279 table I a) or a variant thereof having proteolytic activity.

Preparation of enzyme preparations

The present invention relates to a process for preparing an enzyme preparation, said process comprising at least the step of mixing component (a) as disclosed above and component (b) as disclosed above.

In one embodiment, the present invention relates to a process for preparing an enzyme preparation, said process comprising the steps of mixing components (a), (b) and (c) as disclosed above, wherein component (b) preferably comprises at least one amylase; and optionally at least one enzyme selected from the group consisting of proteases, lipases, cellulases and mannanases. The amylase is preferably selected from the group of alpha-amylases as disclosed above (EC 3.2.1.1), more preferably at least one amylase selected from the group consisting of:

In one embodiment, component (c) comprises at least one solvent as disclosed above. In one embodiment, component (c) comprises at least one enzyme stabilizer different from component (a) as disclosed above.

Component (b) may be a solid. The solid component (b) may be added to the solid component (a) before both are contacted with at least one solvent (component (c)). The at least one solvent is as disclosed above. The contact with the at least one solvent (component (c)) may lead to a solubilization of the at least one molecular component (a) and the at least one molecular component (b), thus leading to a stabilization of the at least one molecular component (b). In one embodiment, the solid components (a) and (b) are completely dissolved in the at least one solvent (component (c)) without phase separation.

The solid component (a) may be dissolved in at least one solvent (component (c)) before mixing with the solid or liquid component (b). In one embodiment, component (a) is completely dissolved in at least one solvent (component (c)) prior to mixing with component (b). The at least one solvent is as disclosed above.

Component (b) may be a liquid, wherein the at least one enzyme may be comprised in a liquid enzyme concentrate as disclosed above. The liquid component (b) may be supplemented with the solid component (a), wherein the solid component (a) is dissolved in the liquid component (b). In one embodiment, the liquid component (b) is aqueous, preferably obtained by fermentation. In one embodiment, when the solid component (a) is dissolved in the liquid component (b), no additional solvent may be added.

In one embodiment, component (c) as disclosed above is mixed with components (a) and (b), wherein the mixing is characterized by being carried out in one or more steps.

Enzyme stabilization

The present invention relates to a process for stabilizing at least one hydrolase contained in component (b) by the step of adding component (a), wherein components (a) and (b) are those disclosed above. In one embodiment, component (b) is a liquid. In one embodiment, the present invention relates to a method for stabilizing component (b) by the step of adding component (a), wherein component (b) comprises at least one amylase and/or at least one protease and/or at least one lipase and/or at least one mannanase.

The at least one amylase may be selected from the group of alpha-amylases as disclosed above (EC 3.2.1.1), more preferably the at least one amylase is selected from the group consisting of:

The at least one protease may be selected from subtilisin-type proteases (EC 3.4.21.62), preferably from:

At least one lipase may be a Thermomyces lanuginosus lipase selected from variants having lipolytic activity, which variants are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or identical when compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO. 2 of US 5869438. Preferably, the Thermomyces lanuginosus lipases comprise only conservative mutations, but they do not relate to the functional domain of amino acids 1-269 of SEQ ID NO:2 of US 5869438. The Thermomyces lanuginosus lipase may be characterized by having at least the amino acid substitutions T231R and N233R in SEQ ID NO 2 of US 5869438.

In one embodiment, the present invention relates to a process for stabilizing component (b) by the addition of component (a) and at least one enzyme stabilizer different from component (a) as disclosed above, preferably an enzyme stabilizer (component (c)) selected from polyols, peptide aldehydes and other stabilizers as disclosed above.

In one embodiment, the present invention relates to a method of stabilizing component (b) in the presence of at least one surfactant by the step of adding component (a) and optionally at least one enzyme stabilizer different from component (a) as disclosed above, wherein components (a) and (b) are those disclosed above and the at least one surfactant is selected from the group consisting of nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants, all as described below. In one embodiment, the liquid formulation is a detergent formulation.

In one embodiment, the present invention relates to the use of a compound of formula (I), component (a) as disclosed above, as an additive to at least one hydrolase (component (b)):

wherein the variables in formula (I) are defined as follows:

R¹selected from H and C₁-C₁₀Alkylcarbonyl, wherein the alkyl may be linear or branched and may carry one or more hydroxyl groups;

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R ⁴At least one of which is not H;

wherein the compound of formula (I) and the hydrolase are solids and wherein the enzymatic activity of the hydrolase is stabilized upon contacting the compound of formula (I) and the hydrolase with at least one solvent [ component (c) ].

The contact with at least one solvent (component (c)) may lead to a solubilization of the at least one molecular component (a) and the at least one molecular component (b), thus leading to a stabilization of the at least one molecular component (b). In one embodiment, the solid components (a) and (b) are completely dissolved in the at least one solvent (component (c)) without phase separation.

In one embodiment of the invention, component (a) is added in an amount in the range of 0.1 to 30% by weight relative to the total weight of the enzyme preparation. The enzyme preparation may comprise component (a) in an amount in the range of 0.1-15 wt. -%, 0.25-10 wt. -%, 0.5-6 wt. -% or 1-3 wt. -%, all relative to the total weight of the enzyme preparation.

In one embodiment, the at least one enzyme stabilizer different from component (a) is added in an amount effective for reversibly inhibiting the proteolytic activity of the at least one protease comprised in component (b).

In one embodiment, said compound of formula (I) (component (a)) is used as an additive to component (b), wherein component (b) comprises at least one amylase preferably selected from the group consisting of alpha-amylases (EC 3.2.1.1) as disclosed above, wherein the compound of formula (I) and the amylase are solid and wherein the amylolytic activity of the amylase is stabilized upon contact of the compound of formula (I) and the amylase with at least one solvent [ component (c) ]. The amylase may be selected from:

In one embodiment, component (b) comprises at least one protease selected from subtilisin-type proteases (EC 3.4.21.62), preferably selected from:

In one embodiment, component (b) comprises at least one amylase and at least one protease, wherein the at least one protease may be selected from subtilisin-type proteases (EC 3.4.21.62), preferably from:

In one embodiment, component (b) comprises at least one lipase selected from Thermomyces lanuginosus lipase and variants thereof having lipolytic activity, which variants are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438. Preferably, the Thermomyces lanuginosus lipases comprise only conservative mutations, but they do not relate to the functional domain of amino acids 1-269 of SEQ ID NO:2 of US 5869438. The Thermomyces lanuginosus lipase may be characterized by having at least the amino acid substitutions T231R and N233R in SEQ ID NO 2 of US 5869438.

In one embodiment, component (b) comprises at least one amylase and/or at least one protease and/or at least one lipase as disclosed above.

In one embodiment, the compound of formula (I) (component (a)) is used as an additive to a composition comprising at least one hydrolase (component (b)) and at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed herein. The addition of component (a) to component (b) makes it possible to stabilize at least one hydrolase during storage in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably contained in an amount in the range from 1 to 5% by weight, more preferably from 1.5 to 2% by weight, both relative to the total weight of the composition, and/or wherein at least one hydrolase is preferably contained in an amount in the range from 0.2 to 2% by weight, more preferably about 0.5% by weight, both relative to the total weight of the composition, and/or wherein optionally in an amount in the range from 10 to 30% by weight, preferably from 15 to 25%:

EDTA and/or DTPA and/or

MGDA and/or GLDA, all relative to the total weight of the composition. Known as alkaline earth metal ions such as Ca²⁺And Mg²⁺MGDA (methylglycinediacetic acid) and GLDA (glutamic diacetic acid) of the sequestering agents of (a) are those disclosed below.

The at least one hydrolase may be selected from amylases, proteases, lipases, and mannanases-all as disclosed above.

Component (b) in this connection preferably comprises at least one amylase, preferably selected from the group consisting of the alpha-amylases (EC 3.2.1.1) as disclosed above, wherein the compound of formula (I) and the amylase are solid and wherein the amylolytic activity of the amylase is stabilized upon contact of the compound of formula (I) and the amylase with at least one solvent [ component (c) ]. The amylase may be selected from:

In one embodiment, said compound of formula (I) is used as an additive to component (b) together with at least one enzyme stabilizer different from component (a), wherein component (b) comprises at least one amylase preferably selected from the group consisting of alpha-amylases (EC 3.2.1.1) as disclosed above, wherein the compound of formula (I), the enzyme stabilizer different from component (a) and the amylase are solid and wherein the amylolytic activity of the amylase is stabilized upon contacting the solid component with at least one solvent [ component (c) ]. The amylase may be selected from:

In one embodiment, component (b) comprises at least one amylase and at least one other enzyme selected from the group consisting of:

a protease selected from serine endopeptidases (EC 3.4.21), preferably from subtilisin-type proteases (EC 3.4.21.62), more preferably from a protease according to SEQ ID NO:22 as described in EP 1921147 or a variant thereof having proteolytic activity as disclosed above and from subtilisin 309 or a variant thereof having proteolytic activity as disclosed above in Table I a of WO 89/06279, and

a lipase selected from the group of triacylglycerol lipases, preferably from the group of triacylglycerol lipases of amino acids 1 to 269 of SEQ ID NO:2 according to US 5869438 or variants thereof having lipolytic activity as disclosed above,

and optionally at least one enzyme stabilizer different from component (a), preferably an enzyme stabilizer selected from the group consisting of boron-containing compounds, polyols, peptide aldehydes and other stabilizers as disclosed above,

wherein all components are solid and wherein the amylolytic activity of the amylase and/or the proteolytic activity of the protease and/or the lipolytic activity of the lipase is stabilized upon contacting the components with at least one solvent [ component (c) ].

Stabilization of an enzyme may involve stability over time (e.g., storage stability), thermal stability, pH stability, and chemical stability. The term "enzyme stability" herein preferably relates to the retention of enzymatic activity, e.g. over time during storage or handling. The term "storage" is intended herein to mean the fact that the product or composition is stored from the time it is prepared to the point in time it is used in the final application. The retention of enzymatic activity as a function of time during storage is referred to as "storage stability". In one embodiment, storage refers to storage at 37 ℃ for at least 20 days. Storage may refer to storage at 37 ℃ for 21, 28 or 42 days.

To determine the change in enzymatic activity over time, the "initial enzymatic activity" of an enzyme may be measured at time zero (i.e. before storage) and the "enzymatic activity after storage" may be measured at some later point in time (i.e. after storage) under defined conditions.

The enzymatic activity after storage is divided by the initial enzymatic activity multiplied by 100 to give the "residual enzymatic activity" (a%).

It is stable according to the invention when the residual enzymatic activity of the enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% compared to the initial enzymatic activity before storage.

Subtracting a% from 100% gives the "loss of enzymatic activity during storage" when compared to the initial enzymatic activity prior to storage. In one embodiment, the enzyme according to the invention is stable when substantially no loss of enzymatic activity occurs during storage, i.e. the loss of enzymatic activity is equal to 0% when compared to the initial enzymatic activity prior to storage. Substantially no loss of enzymatic activity in the context of the present invention may mean a loss of enzymatic activity of less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% when compared to the initial enzymatic activity prior to storage.

In one aspect of the invention, component (a) is used to reduce the loss of enzymatic activity during storage of component (b). The calculation of the% reduction in enzymatic activity loss was performed as follows: (% loss of enzymatic Activity of stabilized enzyme) - (% loss of enzymatic Activity of unstabilized enzyme). The loss reduction value shows that the loss of enzymatic activity of at least one enzyme comprised in component (b) in the presence of component (a) is reduced when compared to the loss of enzymatic activity of the same enzyme in the absence of component (a) at a certain point in time.

A reduction in the loss of enzymatic activity in the present invention may mean a reduction of the loss of enzymatic activity in the presence of component (a) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% when compared to the loss of enzymatic activity in the absence of component (a).

One aspect of the present invention relates to a method for reducing the loss of amylolytic activity during storage of at least one amylase contained in a liquid, preferably selected from the group consisting of alpha-amylases contained in component (b), by the step of adding a compound of formula (I):

Wherein the variables in formula (I) are defined as follows:

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H.

In one embodiment, the method of reducing the loss of amylolytic activity of at least one amylase contained in a liquid (component (b)) during storage comprises the steps of adding a compound of formula (I) and adding at least one enzyme stabilizer different from component (a), preferably an enzyme stabilizer selected from the group consisting of polyols, peptide aldehydes and other stabilizers as disclosed above.

In one embodiment, the amylase (component (b)) is comprised in a liquid enzyme formulation, or the amylase is comprised in a liquid composition comprising at least one surfactant, such as a liquid detergent formulation, the latter preferably further comprising at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed herein, which amylase may be selected from alpha-amylases as disclosed above (EC 3.2.1.1), preferably selected from the following alpha-amylases:

A hybrid amylase according to WO 2014/183921, wherein the A and B domains have at least 75% identity to SEQ ID NO 2, SEQ ID NO 15, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 32 and SEQ ID NO 39 and the C domain has at least 90% identity to SEQ ID NO 6 of WO 2014/183921 as disclosed in WO 2014/183921, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO 30 as disclosed in WO 2014/183921 and has amylolytic activity. In one embodiment, component (b) comprises at least one amylase preferably selected from the group consisting of the alpha-amylases disclosed above (EC 3.2.1.1), preferably selected from the group consisting of

In one embodiment, component (b) comprises at least one amylase and at least one protease selected from serine endopeptidases (EC 3.4.21), preferably from subtilisin-type proteases (EC 3.4.21.62), more preferably from proteases according to SEQ ID NO:22 as described in EP 1921147 or variants thereof having proteolytic activity as disclosed above and from subtilisin 309 or variants thereof having proteolytic activity as disclosed above in WO 89/06279 table I a).

In one aspect of the invention, component (a) stabilizes at least one amylase comprised in component (b). The at least one amylase comprised in component (b) is preferably selected from the group of alpha-amylases disclosed above (EC 3.2.1.1). In one embodiment, component (a) is used to stabilize amylase [ component (b) ] in a liquid enzyme formulation. In one embodiment, component (a) is used to stabilize amylase in a liquid composition comprising at least one surfactant, preferably in a liquid detergent composition [ component (b) ]. Stabilization may in this connection mean stabilization during storage at 37 ℃ for 21, 28 and/or 42 days.

In one embodiment, adding component (a) to component (b) stabilizes the amylase during storage, wherein the stabilization is characterized by:

(a) the residual amylolytic activity after 21 days of storage at 37 ℃ is more than or equal to 60%, more than or equal to 70%, more than or equal to 80% or more than or equal to 90% when compared to the initial amylolytic activity prior to storage, and/or

(b) Residual amylolytic activity after 28 days of storage at 37 ℃ is ≥ 55%, ≥ 60%, ≥ 70% or ≥ 80%, and/or

(c) The residual amylolytic activity after 42 days of storage at 37 ℃ is more than or equal to 35%, more than or equal to 45%, more than or equal to 50% or more than or equal to 60% when compared to the initial amylolytic activity prior to storage.

(d)

Addition of component (a) to component (b) may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably comprised in an amount in the range of 1-5 wt.%, more preferably 1.5-2 wt.%, both relative to the total weight of the composition, and/or wherein the amylase is preferably comprised in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, both relative to the total weight of the composition, and/or wherein optionally the amylase is comprised in an amount in the range of 10-30 wt.%, preferably 15-25 wt.%:

EDTA and/or DTPA and/or

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, and wherein the stabilization is characterized by:

(a) residual amylolytic activity after 21 days of storage at 37 ℃ is > 75%, > 80% or > 85% when compared to the initial amylolytic activity prior to storage, and/or

(b) Residual amylolytic activity after 28 days of storage at 37 ℃ is more than or equal to 65%, more than or equal to 70% or more than or equal to 80% when compared to the initial amylolytic activity prior to storage, and/or

(c) The residual amylolytic activity after 42 days of storage at 37 ℃ is more than or equal to 55%, more than or equal to 60% or more than or equal to 65% when compared to the initial amylolytic activity prior to storage.

(d)

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl radicals, and

wherein component (a) is preferably included in an amount in the range of from 1 to 5% by weight, more preferably from 1.5 to 2% by weight, each relative to the total weight of the composition, and/or

Wherein preferably the amylase is comprised in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, all relative to the total weight of the composition, and/or

Wherein optionally:

-EDTA and/or DTPA is contained in an amount of at most 3% by weight, preferably at most 2.5% relative to the total weight of the composition, all relative to the total weight of the composition, and/or

MGDA and/or GLDA is contained in an amount ranging from 10 to 30% by weight, preferably from 15 to 25%, all relative to the total weight of the composition.

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl, and wherein the stabilization is characterized by:

(a) residual amylolytic activity after 21 days of storage at 37 ℃ is more than or equal to 65%, more than or equal to 70% or more than or equal to 80% when compared to the initial amylolytic activity prior to storage, and/or

(b) Residual amylolytic activity after 28 days of storage at 37 ℃ is ≥ 55%, ≥ 65% or ≥ 70% when compared to the initial amylolytic activity prior to storage, and/or

(c) The residual amylolytic activity after 42 days of storage at 37 ℃ is more than or equal to 35%, more than or equal to 50% or more than or equal to 60% when compared to the initial amylolytic activity prior to storage.

(d)

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl groups, and wherein component (a) is preferably included in an amount in the range of from 1 to 5 wt.%, more preferably from 1.5 to 2 wt.%, each relative to the total weight of the composition, and/or

Wherein optionally:

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(a) the residual amylolytic activity after 21 days of storage at 37 ℃ is more than or equal to 60%, more than or equal to 70% or more than or equal to 75% when compared to the initial amylolytic activity prior to storage, and/or

(b) Residual amylolytic activity after 28 days of storage at 37 ℃ is ≥ 55%, ≥ 60% or ≥ 65% and/or

(c) The residual amylolytic activity after 42 days of storage at 37 ℃ is more than or equal to 45%, more than or equal to 50% or more than or equal to 60% when compared to the initial amylolytic activity prior to storage.

(d)

Addition of component (a) to component (b) possibly stabilizing the amylase during storage in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein component (a) is preferably included in an amount in the range of from 1 to 5% by weight, more preferably from 1.5 to 2% by weight, each relative to the total weight of the composition, and/or

Wherein optionally:

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of phenylmethyl and salicyl, and The customization is characterized in that:

residual amylolytic activity after 21 days of storage at 37 ℃ of 65% or more, 75% or more or 80% or more and/or

Residual amylolytic activity after 28 days of storage at 37 ℃ of ≥ 60%, > 65%, > 70% or ≥ 80% when compared to the initial amylolytic activity prior to storage, and/or

Residual amylolytic activity after 42 days of storage at 37 ℃ is ≥ 55%, ≥ 60% or ≥ 70% when compared to the initial amylolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is H and R²、R³、R⁴Selected from phenylmethyl and salicyl, and

Wherein optionally:

(a) the loss of amylolytic activity during storage at 37 ℃ for 21 days is 35% or less, 30% or 25% or less, and/or

(b) The loss of amylolytic activity during storage at 37 ℃ for 28 days is less than or equal to 40%, less than or equal to 35% or less than or equal to 30% when compared to the initial amylolytic activity prior to storage, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days is less than or equal to 60%, less than or equal to 50% or less than or equal to 45% when compared to the initial amylolytic activity prior to storage.

Addition of component (a) to component (b) may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably comprised in an amount in the range of 1-5 wt.%, more preferably 1.5-2 wt.%, both relative to the total weight of the composition, and/or wherein the amylase is preferably comprised in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, both relative to the total weight of the composition, and/or wherein optionally:

(a) a loss of amylolytic activity during storage at 37 ℃ for 21 days of ≤ 20% or ≤ 5% when compared to the initial amylolytic activity before storage, and/or

(b) A loss of amylolytic activity during storage at 37 ℃ for 28 days of 31% or 25% or less when compared to the initial amylolytic activity prior to storage, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days is 43% or 35% or less when compared to the initial amylolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA ¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl radicals, and

Wherein optionally:

(a) a loss of amylolytic activity during storage at 37 ℃ for 21 days of 32% or 5% or less when compared to the initial amylolytic activity prior to storage, and/or

(b) A loss of amylolytic activity during storage at 37 ℃ for 28 days of less than or equal to 40% or less than or equal to 25% when compared to the initial amylolytic activity prior to storage, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days is less than or equal to 60%, less than or equal to 50% or less than or equal to 40% when compared to the initial amylolytic activity prior to storage.

Mixing component (a)) Addition to component (b), which may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, where component (a) is characterized by R in the compound of formula (I)¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl groups, and wherein component (a) is preferably included in an amount in the range of from 1 to 5 wt.%, more preferably from 1.5 to 2 wt.%, each relative to the total weight of the composition, and/or

Wherein optionally:

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I) ¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(a) the loss of amylolytic activity during storage at 37 ℃ for 21 days is < 34%, < 27% or < 5%, and/or

(b) The loss of amylolytic activity during storage at 37 ℃ for 28 days is < 42% or < 36 when compared to the initial amylolytic activity prior to storage, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days is < 50% or < 46% when compared to the initial amylolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein component (a) is preferably included in an amount in the range of from 1 to 5% by weight, more preferably from 1.5 to 2% by weight, each relative to the total weight of the composition, and/or

Wherein optionally:

·

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of phenylmethyl and salicyl, and wherein the stabilization is characterized by:

(a) the loss of amylolytic activity during storage at 37 ℃ for 21 days is < 31%, < 20%, < 15% or < 5%, and/or

(b) The loss of amylolytic activity during storage at 37 ℃ for 28 days is < 37%, < 30% or < 25% or < 20% when compared to the initial amylolytic activity prior to storage, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days is < 43%, < 35% or < 30% when compared to the initial amylolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA ¹Is H and R²、R³、R⁴Selected from phenylmethyl and salicyl, and

Wherein optionally:

(a) the reduction in amylolytic activity loss during storage at 37 ℃ for 21 days is ≥ 14% when compared to the amylolytic activity loss in the absence of component (a), and/or

(b) The reduction in amylolytic activity loss during 28 days of storage at 37 ℃ when compared to the amylolytic activity loss in the absence of component (a) is ≥ 29%, and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days was reduced by > 24% when compared to the loss of amylolytic activity in the absence of component (a).

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of linear C ₂-C₄Alkyl, and wherein the stabilization is characterized by:

(a) the reduction of the loss of amylolytic activity during storage at 37 ℃ for 21 days is ≥ 14%, > 25% or ≥ 40%, and/or

(b) The loss of amylolytic activity during storage at 37 ℃ for 28 days is reduced by ≥ 40% when compared to the loss of amylolytic activity in the absence of component (a), and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days was reduced by ≥ 37% when compared to the loss of amylolytic activity in the absence of component (a).

Wherein optionally:

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl, and wherein the stabilization is characterized by: (a) loss of amylolytic Activity during storage at 37 ℃ for 21 days compared to absence of component (a)

A reduction of 18% or 30% or more when compared to the loss of amylolytic activity, and/or (b) a loss of amylolytic activity during 28 days of storage at 37 ℃ when compared to the loss of amylolytic activity in the absence of component (a)

A reduction of 33% or 40% or more when compared to the loss of amylolytic activity, and/or (c) a loss of amylolytic activity during storage at 37 ℃ for 42 days when compared to the loss of amylolytic activity in the absence of component (a)

The reduction in the loss of amylolytic activity is greater than or equal to 23% or greater than or equal to 30% when compared.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), may stabilize the amylase during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl, and preferably in the range of 1-5% by weightMore preferably in the range of from 1.5 to 2% by weight, comprises component (a), all relative to the total weight of the composition, and/or

Wherein optionally:

In one embodiment, addition of component (a) to component (b) stabilizes the amylase during storage, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C ₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(a) the loss of amylolytic activity during storage at 37 ℃ for 21 days is reduced by more than or equal to 16% or more than or equal to 25% when compared to the loss of amylolytic activity in the absence of component (a), and/or

Wherein optionally:

(a) the loss of amylolytic activity during storage at 37 ℃ for 21 days is reduced by more than or equal to 29% or more than or equal to 40% when compared to the loss of amylolytic activity in the absence of component (a), and/or

(b) The loss of amylolytic activity during storage at 37 ℃ for 28 days is reduced by 34% or 40% or more when compared to the loss of amylolytic activity in the absence of component (a) and/or

(c) The loss of amylolytic activity during storage at 37 ℃ for 42 days was reduced by > 38% when compared to the loss of amylolytic activity in the absence of component (a).

Wherein optionally:

In an embodiment of the above embodiment, component (a) as disclosed above is used to stabilize amylase [ component (b) ] in a liquid enzyme preparation. Furthermore, in an embodiment of the above embodiment, the amylase stabilized by component (a) is an alpha-amylase as disclosed above (EC 3.2.1.1), preferably an alpha-amylase selected from the group consisting of:

In one aspect of the invention, component (a) is used for stabilizing component (b) in a liquid composition preferably comprising at least one surfactant and/or at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed herein, the latter comprising at least one amylase, preferably an alpha-amylase as disclosed above (EC 3.2.1.1), and at least one protease, preferably a protease selected from serine endopeptidases (EC 3.4.21), more preferably from subtilisin type proteases (EC 3.4.21.62), wherein the at least one amylase is selected from:

and wherein the at least one protease is selected from subtilisin-type proteases (EC 3.4.21.62); preferably selected from the group consisting of the protease according to SEQ ID NO:22 as described in EP 1921147 or a proteolytically active variant thereof as disclosed above and from the group consisting of subtilisin 309 as disclosed in WO 89/06279 Table I a) or a proteolytically active variant thereof as disclosed above.

In one aspect of the invention, component (a) stabilizes at least one protease comprised in component (b). At least one protease is comprised in component (b), preferably selected from subtilisin-type proteases as disclosed above (EC 3.4.21.62). In one embodiment, component (a) is used to stabilize the protease [ component (b) ] in a liquid enzyme formulation. In one embodiment, component (a) is used to stabilize a protease in a liquid composition comprising at least one surfactant and/or at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed herein [ component (b) ]. Stabilization may in this connection mean stabilization during storage at 37 ℃ for 14, 21, 28 and/or 42 days.

In one embodiment, adding component (a) to component (b) stabilizes the protease during storage, wherein the stabilization is characterized by:

(a) residual proteolytic activity after 21 days of storage at 37 ℃ is ≥ 70% or ≥ 80% when compared to the initial proteolytic activity prior to storage, and/or

(b) Residual proteolytic activity after 28 days of storage at 37 ℃ is ≥ 65%, > 70% or ≥ 75% when compared to the initial proteolytic activity prior to storage, and/or

(c) The residual proteolytic activity after 42 days of storage at 37 ℃ is ≥ 55%, ≥ 60% or ≥ 65% when compared to the initial proteolytic activity prior to storage.

The addition of component (a) to component (b) makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably contained in an amount in the range of 1-5 wt.%, more preferably 1.5-2 wt.%, both relative to the total weight of the composition, and/or wherein the protease is preferably contained in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, both relative to the total weight of the composition, and/or wherein optionally:

MGDA and/or GLDA is contained in an amount ranging from 10 to 30% by weight, preferably from 15 to 25%, all relative to the total weight of the composition. Known as alkaline earth metal ions such as Ca²⁺And Mg²⁺MGDA (methylglycinediacetic acid) and GLDA (glutamic diacetic acid) of the sequestering agents of (a) are those disclosed below.

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I) ¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl radical ofAnd wherein the stabilization is characterized by:

(a) residual proteolytic activity after 21 days of storage at 37 ℃ is > 75% or > 80% when compared to the initial proteolytic activity prior to storage, and/or

(b) Residual proteolytic activity after 28 days of storage at 37 ℃ is ≥ 70% or ≥ 75% when compared to the initial proteolytic activity prior to storage, and/or

(c) The residual proteolytic activity after 42 days of storage at 37 ℃ is ≥ 65% when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is H and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl radicals, and

Wherein the protease is preferably included in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, all relative to the total weight of the composition, and/or

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl, and wherein the stabilization is characterized by:

(c) The residual proteolytic activity after 42 days of storage at 37 ℃ is ≥ 60% or ≥ 65% when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C ₂And C₄Alkyl groups, and wherein component (a) is preferably included in an amount in the range of from 1 to 5 wt.%, more preferably from 1.5 to 2 wt.%, each relative to the total weight of the composition, and/or

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(a) residual proteolytic activity after 21 days of storage at 37 ℃ is ≥ 70% or ≥ 75% when compared to the initial proteolytic activity prior to storage, and/or

(b) Residual proteolytic activity after 28 days of storage at 37 ℃ is ≥ 65% or ≥ 70% when compared to the initial proteolytic activity prior to storage, and/or

(c) The residual proteolytic activity after 42 days of storage at 37 ℃ is ≥ 55% or ≥ 60% when compared to the initial proteolytic activity prior to storage.

The addition of component (a) to component (b), which may stabilize the protease during storage in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And an

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I) ¹Is H and R²、R³、R⁴Selected from the group consisting of phenylmethyl and salicyl, and wherein the stabilization is characterized by:

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is H and R²、R³、R⁴Selected from phenylmethyl and salicyl, and

Wherein optionally:

(a) a loss of proteolytic activity during storage at 37 ℃ for 21 days of 27% or 20% or less, and/or

(b) The loss of proteolytic activity during 28 days of storage at 37 ℃ is 33% or 23% or less when compared to the initial proteolytic activity prior to storage, and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is < 44% or < 38% when compared to the initial proteolytic activity prior to storage.

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H, and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, and wherein the stabilization is characterized by:

(a) a loss of proteolytic activity during storage at 37 ℃ for 21 days of ≦ 20% when compared to the initial proteolytic activity prior to storage, and/or

(b) A loss of proteolytic activity during 28 days of storage at 37 ℃ of < 28% when compared to the initial proteolytic activity prior to storage, and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is < 33% when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA ¹Is H, and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl radicals, and

Wherein optionally:

(a) a loss of proteolytic activity during storage at 37 ℃ for 21 days of < 21% when compared to the initial proteolytic activity prior to storage, and/or

(b) A loss of proteolytic activity during storage at 37 ℃ for 28 days of less than or equal to 29% or less than or equal to 25% when compared to the initial proteolytic activity prior to storage, and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is < 35% when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹Is acetyl and R²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂And C₄Alkyl radicals, and

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I) ¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(a) a loss of proteolytic activity during storage at 37 ℃ for 21 days of 27% or 23% or less, and/or

(b) A loss of proteolytic activity during storage at 37 ℃ for 28 days of < 33% when compared to the initial proteolytic activity prior to storage, and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is 45% or 40% or less when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C₂Alkyl and R³Is equal to R¹/R²Or R⁴And an

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R²、R³、R⁴Selected from the group consisting of phenylmethyl and salicyl, and wherein the stabilization is characterized by:

(a) a loss of proteolytic activity during storage at 37 ℃ for 21 days of less than or equal to 21% or less than or equal to 15% when compared to the initial proteolytic activity prior to storage, and/or

(b) A loss of proteolytic activity during storage at 37 ℃ for 28 days of less than or equal to 30% or less than or equal to 25% when compared to the initial proteolytic activity prior to storage, and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is 36% or 30% or less when compared to the initial proteolytic activity prior to storage.

Addition of component (a) to component (b), which component (a) is characterized by R in the compound of formula (I), makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA ¹Is H and R²、R³、R⁴Selected from phenylmethyl and salicyl, and

Wherein optionally:

(a) the loss of proteolytic activity during storage at 37 ℃ for 21 days is reduced by more than or equal to 5% or more than or equal to 10% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(b) The loss of proteolytic activity during storage at 37 ℃ for 28 days is reduced by more than or equal to 5% or more than or equal to 10% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is reduced by 5% or more or 10% or more when compared to the loss of proteolytic activity in the absence of component (a).

Addition of component (a) to component (b) makes it possible to stabilize the protease during storage, preferably in the presence of complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably contained in an amount in the range of 1-5 wt.%, more preferably 1.5-2 wt.%, both relative to the total weight of the composition, and/or wherein the protease is preferably contained in an amount in the range of 0.2-2 wt.%, more preferably about 0.5 wt.%, both relative to the total weight of the composition, and/or

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹Is H and R ²、R³、R⁴Selected from the group consisting of linear C₂-C₄Alkyl, and wherein the stabilization is characterized by:

(a) the loss of proteolytic activity during storage at 37 ℃ for 21 days is reduced by more than or equal to 5%, more than or equal to 10% or more than or equal to 14% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(b) The loss of proteolytic activity during storage at 37 ℃ for 28 days is reduced by more than or equal to 2%, more than or equal to 5%, more than or equal to 10% or more than or equal to 20% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is reduced by more than or equal to 10%, more than or equal to 15% or more than or equal to 20% when compared to the loss of proteolytic activity in the absence of component (a).

Wherein optionally:

·

(a) the loss of proteolytic activity during storage at 37 ℃ for 21 days is reduced by more than or equal to 5%, more than or equal to 10% or more than or equal to 15% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(b) The loss of proteolytic activity during storage at 37 ℃ for 28 days is reduced by more than or equal to 5%, more than or equal to 10% or more than or equal to 20% when compared to the loss of proteolytic activity in the absence of component (a), and/or

(c) The loss of proteolytic activity during storage at 37 ℃ for 42 days is reduced by not less than 9%, not less than 15% or not less than 20% when compared to the loss of proteolytic activity in the absence of component (a).

Wherein optionally:

In one embodiment, component (a) is added to component (b) to stabilize the protease during storage, wherein component (a) is characterized by R in the compound of formula (I)¹And R²Is H, R⁴Selected from the group consisting of linear C₂-C₄Alkyl, preferably C ₂Alkyl and R³Is equal to R¹/R²Or R⁴And wherein the stabilization is characterized by:

(b) The loss of proteolytic activity during storage at 37 ℃ for 28 days is reduced by more than or equal to 2%, more than or equal to 5% or more than or equal to 10% when compared to the loss of proteolytic activity in the absence of component (a), and/or

Wherein optionally:

(a) the loss of proteolytic activity during storage at 37 ℃ for 21 days is reduced by 4% or 10% or more when compared to the loss of proteolytic activity in the absence of component (a) and/or

Wherein optionally:

In embodiments, the subtilisin-type protease (EC 3.4.21.62) of the above embodiments may be selected from the protease according to SEQ ID NO:22 as described in EP 1921147 or a variant thereof having proteolytic activity as disclosed above and from subtilisin 309 as disclosed in WO 89/06279 table I a) or a variant thereof having proteolytic activity as disclosed above.

Use of enzyme preparations in a method of formulation

In one aspect the present invention relates to the use of the liquid enzyme preparations of the present invention to be formulated into detergent formulations, such as I & I and home care formulations, for laundry and hard surface cleaning, wherein at least components (a) and (b) are mixed with one or more detergent components in one or more steps in a non-specified order. In one embodiment, at least components (a), (b) and (c) as disclosed above are mixed with one or more detergent components in a non-specified order in one or more steps.

In one aspect, the present invention relates to a detergent formulation comprising a liquid enzyme preparation of the invention and one or more detergent components.

The detergent components may vary in type and/or amount in the detergent formulation depending on the desired application, such as washing white textiles, colored textiles and wool. The components selected further depend on the physical form of the detergent formulation (liquid, solid, gel, provided in a sachet or as a tablet, etc.). The choice of components for laundry formulations, for example, is further dependent on the regional habit, the latter itself being related to aspects and geographical features such as the washing temperature used, the washing machine machinery (drum washing machine versus drum washing machine), the amount of water used per wash cycle, etc., such as the average hardness of the water.

The individual detergent components and the amounts used in the detergent formulation are known to the person skilled in the art. Suitable detergent components comprise, inter alia, surfactants, builders, polymers, alkalis, bleaching systems, optical brighteners, suds suppressors and stabilizers, hydrotropes and corrosion inhibitors. Other examples are described, for example, in "complete Technology Book on Detergents with Formulations (Detergents Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray driven Powder)", Engineers India Research Institute (EIRI), 6 th edition (2015). Another reference book may be "reagent formulas Encyclopedia", Solverchem Publications, 2016, to those skilled in the art.

It is to be understood that the detergent component furthermore also comprises components which are contained in the enzyme preparation of the invention. If the component contained in the enzyme preparation of the invention is also a detergent component, it may be a concentrate which needs to be adjusted so that this component is effective for the desired purpose in the detergent formulation.

The detergent component may have more than one function in the end use of the detergent formulation and thus any detergent component mentioned herein with respect to a particular function may also have another function in the end use of the detergent formulation. The function of a particular detergent component in the end use of a detergent formulation is generally dependent on its amount in the detergent formulation, i.e. the effective amount of the detergent component.

The term "effective amount" includes an amount of an individual component that provides effective stain removal and/or effective cleaning conditions (e.g., pH, foaming amount), an amount of some component that is effective to provide an optical benefit (e.g., fluorescence whitening, dye transfer inhibition) and/or an amount of some component that is effective to aid in processing (maintaining physical properties during processing, storage and use; e.g., viscosity modifiers, hydrotropes, desiccants).

In one embodiment, the detergent formulation is a formulation of more than two detergent components, wherein at least one component is effective in detersive, at least one component is effective in providing optimal cleaning conditions and at least one component is effective in maintaining the physical characteristics of the detergent.

The detergent formulations of the invention may comprise component (a) and component (b) dissolved in a solvent. Dissolution may refer to dissolution throughout the detergent formulation. By dissolved may be meant that component (a) and component (b) are part of a liquid enzyme formulation of the invention that may be encapsulated. The encapsulated liquid enzyme preparation may be part of a liquid detergent formulation or part of a solid detergent formulation.

In one embodiment of the present invention, the detergent formulation, preferably the liquid detergent formulation, comprises component (a) in an amount in the range of from 0.1 to 30 wt. -%, relative to the total weight of the detergent formulation. The enzyme preparation may comprise component (a) in an amount in the range of 0.1-15 wt%, 0.25-10 wt%, 0.5-6 wt% or 1-3 wt%, all relative to the total weight of the detergent formulation.

In one embodiment of the present invention, a detergent formulation, preferably a liquid detergent formulation, comprises from 0.5 to 20% by weight, in particular from 1 to 10% by weight, of component (b) and from 0.01 to 10%, more in particular from 0.05 to 5% by weight, most in particular from 0.1 to 2% by weight, of component (a), all relative to the total weight of the detergent formulation.

In one embodiment, the detergent formulation of the invention is liquid at 20 ℃ and 101.3 kPa. The liquid detergent formulation may comprise water or may be substantially free of water, meaning that no significant amount of water is present. By an insignificant amount of water is meant herein that the liquid detergent formulation comprises less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2% by weight of water, all relative to the total weight of the liquid detergent formulation, or is free of water. In one embodiment, an enzyme concentrate that is free of water means that the liquid detergent formulation does not comprise a significant amount of water, but does comprise an organic solvent in an amount of 30-80 wt.%, relative to the total weight of the enzyme concentrate.

Liquid detergent formulations comprising water may comprise water as the sole solvent. In embodiments, a mixture of water and one or more water-miscible solvents is used as the aqueous medium. The term water-miscible solvent relates to an organic solvent which can be miscible with water without phase separation at ambient temperature. Examples are ethylene glycol, 1, 2-propanediol, isopropanol and diethylene glycol. Preferably at least 50% by volume of the corresponding aqueous medium relative to the solvent is water.

The detergent formulations of the present invention comprise at least one compound selected from the group consisting of surfactants, builders, polymers, perfumes and dyes.

The detergent formulations of the present invention comprise at least one surfactant selected from the group consisting of nonionic surfactants, amphoteric surfactants, anionic surfactants and cationic surfactants.

The detergent formulation may comprise 0.1-60 wt% surfactant, relative to the total weight of the detergent formulation. The detergent formulation may comprise at least one compound selected from anionic surfactants, nonionic surfactants, amphoteric surfactants, and amine oxide surfactants, and combinations of at least two of the foregoing. In one embodiment, the detergent formulation of the invention comprises 5-30 wt% anionic surfactant and, for example, in the range of 3-20 wt% of at least one nonionic surfactant, all relative to the total weight of the detergent formulation, wherein the detergent formulation may be a liquid.

The at least one nonionic surfactant may be chosen from alkoxylated alcohols, di-and multiblock copolymers of ethylene oxide and propylene oxide and reaction products of sorbitan with ethylene oxide or propylene oxide, Alkyl Polyglycosides (APG), hydroxyalkyl mixed ethers and amine oxides.

Preferred examples of alkoxylated alcohols and alkoxylated fatty alcohols are, for example, compounds of the general formula (II):

wherein

R³Identical or different and selected from hydrogen and linear C₁-C₁₀Alkyl, preferably identical in each case and being ethyl, particularly preferably hydrogen or methyl,

R⁴selected from branched or linear C₈-C₂₂Alkyl radicals, e.g. n-C₈H₁₇、n-C₁₀H₂₁、n-C₁₂H₂₅、n-C₁₄H₂₉、n-C₁₆H₃₃Or n-C₁₈H₃₇，

R⁵Is selected from C₁-C₁₀Alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1, 2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl or isodecyl.

The variables m and n are in the range from 0 to 300, where the sum of n and m is at least 1, preferably in the range from 3 to 50. Preferably m is in the range of 1-100 and n is in the range of 0-30.

In one embodiment, the compound of formula (II) may be a block copolymer or a random copolymer, preferably a block copolymer.

Further preferred examples of alkoxylated alcohols are, for example, compounds of the general formula (III):

wherein

R⁶Identical or different and selected from hydrogen and linear C₁-C₁₀Alkyl, preferably identical in each case and being ethyl, particularly preferably hydrogen or methyl,

R⁷selected from branched or linear C ₆-C₂₀Alkyl, especially n-C₈H₁₇、n-C₁₀H₂₁、n-C₁₂H₂₅、n-C₁₃H₂₇、n-C₁₅H₃₁、n-C₁₄H₂₉、n-C₁₆H₃₃、n-C₁₈H₃₇，

a is a number in the range from 0 to 10, preferably from 1 to 6,

b is a number in the range from 1 to 80, preferably from 4 to 20,

c is a number in the range from 0 to 50, preferably from 4 to 25.

The sum of a + b + c is preferably in the range of 5 to 100, even more preferably 9 to 50.

In one embodiment, the alkoxylated alcohol is selected from those of formula (III) wherein R is absent⁶And R⁷Is selected from n-C₈H₁₇、n-C₁₀H₂₁、n-C₁₂H₂₅、n-C₁₃H₂₇、n-C₁₅H₃₁、n-C₁₄H₂₉、n-C₁₆H₃₃、n-C₁₈H₃₇(ii) a a and c are 0 and b is in the range of 4 to 20, preferably 9.

Preferred examples of hydroxyalkyl mixed ethers are compounds of the general formula (IV):

wherein the variables are defined as follows:

R⁸identical or different and selected from hydrogen and linear C₁-C₁₀Alkyl, preferably identical in each case and being ethyl, particularly preferably hydrogen or methyl,

R⁹selected from linear or branched C₈-C₂₂Alkyl and C₈-C₂₂An alkenyl group; examples include iso-C₁₁H₂₃iso-C₁₃H₂₇、n-C₈H₁₇、n-C₁₀H₂₁、n-C₁₂H₂₅、n-C₁₄H₂₉、n-C₁₆H₃₃Or n-C₁₈H₃₇，

R¹⁰Selected from linear or branched C₁-C₁₈Alkyl and C₂-C₁₈An alkenyl group; examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1, 2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl, n-dodecyl, n-tetradecyl, n-hexadecyl and n-octadecyl.

The variables m and x are in the range from 0 to 300, preferably from 0 to 100; the sum of m and x is at least 1, preferably in the range from 5 to 50.

The compounds of the formulae (III) and (IV) may be block copolymers or random copolymers, block copolymers being preferred.

Other suitable nonionic surfactants are selected from di-and multiblock copolymers composed of ethylene oxide and propylene oxide. Other suitable nonionic surfactants are selected from ethoxylated or propoxylated sorbitan esters. Amine oxides or alkyl polyglycosides, especially linear C₄-C₁₈Alkyl polyglucosides and branched C₈-C₁₈Alkyl polyglycosides such as compounds of the average formula (V) are likewise suitable.

Wherein:

R¹¹is C₁-C₄Alkyl, especially ethyl, n-propyl or isopropyl,

R¹²is- (CH)₂)₂-R¹¹，

G¹Selected from monosaccharides having 4 to 6 carbon atoms, in particular from glucose and xylose,

y is in the range of 1.1 to 4, where y is an average.

Further examples of nonionic surfactants are compounds of the general formulae (VIa) and (VIb):

wherein

AO is selected from the group consisting of ethylene oxide, propylene oxide and butylene oxide,

EO is oxyethylene CH₂CH₂-O，

R¹³Is C₁-C₄Alkyl, especially ethyl, n-propyl or isopropyl,

R¹⁴selected from branched or linear C₈-C₁₈An alkyl group, a carboxyl group,

A³o is selected from the group consisting of propylene oxide and butylene oxide,

w is a number in the range of 15 to 70, preferably 30 to 50,

w1 and w3 are numbers in the range of 1 to 5, an

w2 is a number in the range of 13-35.

A review of suitable further nonionic surfactants can be found in EP-A0851023 and DE-A19819187.

In one embodiment, the detergent formulation comprises a mixture of two or more different nonionic surfactants.

The at least one amphoteric surfactant may be chosen from surfactants having a positive charge and a negative charge in the same molecule under the conditions of use. A preferred example of an amphoteric surfactant is a so-called betaine surfactant. Many examples of betaine surfactants have one quaternized nitrogen atom and one carboxylic acid group per molecule. A particularly preferred example of an amphoteric surfactant is cocamidopropyl betaine (lauramidopropyl betaine).

Examples of amine oxide surfactants are compounds of the general formula (VII):

R¹³R¹⁴R¹⁵N→O (VII)

wherein R is¹³、R¹⁴And R¹⁵Independently of one another, from aliphatic, cycloaliphatic or C₂-C₄alkylene-C₁₀-C₂₀An alkyl amide moiety. Preferably R¹²Is selected from C₈-C₂₀Alkyl or C₂-C₄alkylene-C₁₀-C₂₀Alkylamido and R¹³And R¹⁴Are all methyl.

A particularly preferred example is lauryl dimethyl amine oxide, sometimes also referred to as laurylamine oxide. Another particularly preferred example is cocamidopropyl dimethyl amine oxide, sometimes also referred to as cocamidopropyl amine oxide.

The at least one anionic surfactant may be chosen from C₈-C₁₈Alkyl sulfuric acid, C₈-C₁₈Fatty alcohol polyether sulfuric acid, ethoxylated C₄-C₁₂Sulfuric acid half esters of alkylphenols (ethoxylation: 1-50mol ethylene oxide/mol), C₁₂-C₁₈Alkyl esters of sulfo fatty acids, e.g. C₁₂-C₁₈Sulfo fatty acid methyl esters, and also C₁₂-C₁₈Alkyl sulfonic acids and C₁₀-C₁₈Alkali metal and ammonium salts of alkylaryl sulfonic acids. Alkali metal salts of the above compounds are preferred, and sodium salts are particularly preferred.

Specific examples of anionic surfactants are compounds of the general formula (VIII):

C_sH_2s+1-O(CH₂CH₂O)_t-SO₃M (VIII)

wherein

s is a number in the range of 10 to 18, preferably 12 to 14, even more preferably s-12,

t is a number in the range of 1 to 5, preferably 2 to 4, even more preferably 3,

m is selected from alkali metals, preferably potassium, even more preferably sodium.

The variables s and t may be averages and thus they need not be integers, whereas in a single molecule of formula (X), both s and t represent integers.

Other examples of suitable anionic surfactants are soaps, such as the sodium or potassium salts of stearic acid, oleic acid, palmitic acid, ether carboxylic acids and alkyl ether phosphoric acids.

The detergent formulations of the invention may comprise one or more compounds selected from complexing agents (chelating agents, sequestering agents), precipitating agents and ion exchange compounds, which may form water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents", but are not intended to limit such compounds to this function in the end use of the detergent formulation.

Non-phosphate based builders of the invention include sodium gluconate, citrate, silicate, carbonate, phosphonate, aminocarboxylate, polycarboxylate, polysulfonate and polyphosphonate.

The detergent formulations of the present invention may comprise one or more citrates. The term "citrate salt" includes mono-and di-alkali metal salts and especially mono-and preferably trisodium salts of citric acid, ammonium or substituted ammonium salts of citric acid and citric acid itself. The citrate may be used as an anhydrous compound or as a hydrate, for example as sodium citrate dihydrate. The detergent formulation of the invention may be comprised in an amount in the range of 0.1-10.0 wt%, 0.5-8.0 wt%, 1.0-5.0 wt% or 2.0-4.0 wt%, all relative to the total weight of the detergent formulation. Citric acid may be provided as a mixture with formate, for example sodium citrate sodium formate 9: 1.

The detergent formulations of the invention may comprise one or more silicates. "silicates" in the context of the present invention include especially sodium disilicate and sodium metasilicate, aluminosilicates such as sodium aluminosilicate, e.g. zeolite A (i.e. Na)₁₂(AlO₂)₁₂(SiO₂)₁₂*27H₂O), and phyllosilicates, in particular of the formula alpha-Na ₂Si₂O₅、β-Na₂Si₂O₅And delta-Na₂Si₂O₅Those of (a).

The detergent formulations of the present invention may comprise one or more carbonates. The term "carbonate" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Sodium carbonate (Na) is particularly preferred₂CO₃)。

The detergent formulations of the present invention may comprise one or more phosphonates. "phosphonates" include, but are not limited to, 2-phosphinobutane-1, 2, 4-tricarboxylic acid (PBT)C) Ethylenediamine tetra (methylenephosphonic acid) (EDTMPA), 1-hydroxyethane-1, 1-diphosphonic acid (HEDP), CH₂C(OH)[PO(OH)₂]₂Amino tri (methylene phosphonic Acid) (ATMP), N [ CH₂PO(OH)₂]₃Amino tri (methylene phosphonic acid) sodium salt (ATMP), N [ CH₂PO(ONa)₂]₃2-hydroxyethyliminodidi (methylenephosphonic acid), HOCH₂CH₂N[CH₂PO(OH)₂]₂Diethylene triamine penta (methylene phosphonic acid) (DTPMP), (HO)₂POCH₂N[CH₂CH₂N[CH₂PO(OH)₂]₂]₂Sodium salt of diethylenetriamine penta (methylenephosphonic acid), C₉H_(28-x)N₃Na_xO₁₅P₅(x ═ 7), potassium hexamethylenediamine (tetramethylenephosphonic acid), and C₁₀H_(28-x)N₂K_xO₁₂P₄(x ═ 6), bis (hexamethylene) triamine (pentamethylenephosphonic acid), (HO)₂)POCH₂N[(CH₂)₂N[CH₂PO(OH)₂]₂]₂. Their salts may also be suitable.

The detergent formulation of the invention may comprise at least one phosphonate, preferably selected from derivatives of polyphosphonic acids such as diphosphonic acids, e.g. the sodium salt of HEDP, derivatives of aminopolyphosphonic acids such as aminoalkylenephosphonic acids, e.g. DTPMP, all in an amount in the range of 0.1-5.0 wt.%, 0.5-3.0 wt.% or 1.0-2.0 wt.%, all relative to the total weight of the detergent formulation.

The detergent formulations of the invention may comprise one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to, diethanol glycine (DEG), dimethyl glycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethyl aminodiacetic acid, Ethylene Diamine Tetraacetic Acid (EDTA), N- (2-hydroxyethyl) iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl ethylenediamine triacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, Diethylene Triamine Pentaacetic Acid (DTPA), Methyl Glycine Diacetic Acid (MGDA), glutamic diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyl imino diacetic acid (IDS)Iminodisuccinic acid, ethylenediamine disuccinic acid (EDDS), aspartic acid diacetic acid and alkali metal or ammonium salts thereof. Further examples are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N, N-diacetic acid (ASDA), aspartic acid-N-monopropionic Acid (ASMP), N- (2-sulfomethyl) aspartic acid (SMAS), N- (2-sulfoethyl) aspartic acid (SEAS), N- (2-sulfomethyl) glutamic acid (SMGL), N- (2-sulfoethyl) glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), alpha-alanine-N, N-diacetic acid (alpha-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, n-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) and sulfomethyl-N, N-diacetic acid (SMDA) and their alkali metal or ammonium salts. The term "ammonium salt" as used in the present context relates to salts having at least one cation with a permanently or temporarily quaternized nitrogen atom. Examples of cations having at least one permanently quaternized nitrogen atom include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium and n-C ₁₀-C₂₀Alkyl trimethyl ammonium. Examples of cations with at least one temporarily quaternized nitrogen atom include protonated amines and ammonia, such as monomethyl ammonium, dimethyl ammonium, trimethyl ammonium, monoethyl ammonium, diethyl ammonium, triethyl ammonium, n-C₁₀-C₂₀Alkyldimethylammonium, 2-hydroxyethylammonium, di (2-hydroxyethyl) ammonium, tri (2-hydroxyethyl) ammonium, N-methyl-2-hydroxyethylammonium, N-dimethyl-2-hydroxyethylammonium and especially NH₄ ⁺。

In one embodiment, the detergent formulation of the present invention comprises more than one builder. Preferably, the detergent formulation of the invention contains less than 0.2 wt% nitrilotriacetic acid (NTA), or 0.01 to 0.1 wt% NTA, relative to the total weight of the detergent formulation.

In one embodiment, the detergent formulation of the invention comprises at least one aminocarboxylate selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycinediacetic acid (MGDA) and glutamic diacetic acid (GLDA), all of which may be (partially) neutralized with a base, in an amount in the range of 0.1 to 25.0 wt. -%, 1.0 to 15.0 wt. -%, 2.0 to 12.0 wt. -% or 2.5 to 10.0 wt. -%, all relative to the total weight of the detergent formulation.

The term base refers to the same or different alkali metal cations, such as cations of lithium, sodium, potassium, rubidium, cesium, and combinations of at least two of the foregoing. Preferred examples of alkali metal cations are sodium and potassium and combinations of sodium and potassium.

In one embodiment, the detergent formulation of the invention comprises at least:

an alkali metal salt of methylglycinediacetic acid (MGDA) in which on average more than 2 and less than 3 carboxyl groups are neutralized by a base, and/or

An alkali metal salt of the L-and D-enantiomers of glutamic diacetic acid (GLDA) or enantiomerically pure L-GLDA, in which on average more than 3 carboxyl groups are neutralized with a base, preferably on average more than 3 and less than 4 carboxyl groups are neutralized with a base.

In one embodiment of the invention, the alkali metal salt of MGDA is selected from compounds of general formula (XIII):

[CH₃-CH(COO)-N(CH₂-COO)₂]M_3-x1-y1(NH₄)_z1H_x1 (XIII)

the variables of formula (XIII) are defined as follows: m is selected from the same or different alkali metal cations, such as cations of lithium, sodium, potassium, rubidium, cesium, and combinations of at least two of the foregoing. Preferred examples of alkali metal cations are sodium and potassium and combinations of sodium and potassium. x1 is selected from 0.0 to 1.0, preferably 0.1 to 0.5, more preferably at most 0.1 to 0.3; z1 is selected from 0.0 to 1.0, preferably 0.0005 to 0.5; however, the sum of x1+ z1 in formula (I) is greater than 0, e.g., 0.05-0.6.

M_3-x1-z1(NH₄)_z1H_x1Is exemplified by Na_3-x1H_x1、[Na_0.7(NH₄)_0.3]_3-x1H_x1、[(NH₄)_0.7Na_0.3]_3-x1H_x1、[(NH₄)_0.7Na_0.3]_3-x1H_x1。

In one embodiment of the invention, the MGDA is selected from at least one alkali metal salt of racemic MGDA and alkali metal salts of a mixture of L-and D-enantiomers of formula (XIII) containing mainly the corresponding L-isomer, wherein the enantiomeric excess (ee) is in the range of 5-99%, preferably 5-95%, more preferably 10-75%, even more preferably 10-66%.

In one embodiment of the invention, the total alkali neutralization degree of MGDA is in the range of 0.80 to 0.98 mol%, preferably 0.90 to 0.97%. The degree of total base neutralization does not take into account any ammonium neutralization.

In one embodiment of the invention, the alkali metal salt of GLDA is selected from compounds of formula (XIV):

[OOC-(CH₂)₂-CH(COO)-N(CH₂-COO)₂]M_4-x2-z2(NH₄)_z2H_x2 (XIV)

the variables of formula (XIV) are defined as follows: m is selected from the same or different alkali metal cations as defined above for formula (XIII); x2 is selected from 0.0 to 2.0, preferably 0.02 to 0.5, more preferably at most 0.1 to 0.3; z2 is selected from 0.0 to 1.0, preferably 0.0005 to 0.5; however, the sum of x2+ z2 in formula (I) is greater than 0, e.g., 0.05-0.6.

M_3-x2-z2(NH₄)_z2H_x1Is exemplified by Na_3-x2H_x2、[Na_0.7(NH₄)_0.3]_3-x2H_x2、[(NH₄)_0.7Na_0.3]_3-x2H_x2。

In one embodiment of the invention, the alkali metal salt of GLDA may be selected from alkali metal salts of the L-and D-enantiomers of formula (XIV), said mixture containing a racemic mixture or preferably mainly the corresponding L-isomer, e.g. with an enantiomeric excess (ee) in the range of 5-99%, preferably 5-95%.

The enantiomeric excess can be determined, for example, by measuring the polarization (polarimetry) or, preferably, by chromatography, for example by HPLC with a chiral column, for example with one or more cyclodextrins as stationary phase or with a ligand exchange (Pirkle brush) notional chiral stationary phase. The enantiomeric excess is preferably determined by HPLC with a fixed optically active ammonium salt, such as D-penicillamine.

In the context of the present invention, small amounts of MGDA and/or GLDA may generally also carry cations other than alkali metals. It is thus possible that minor amounts of builder, e.g. 0.01 to 5 mol% of the total builder, may carry alkaline earth metal cations, e.g. Mg²⁺Or Ca²⁺Or a transition metal cation, e.g. Fe²⁺Or Fe³⁺A cation. "minor amounts" of MGDA and/or GLDA are referred to herein in total of 0.1 to 1% by weight relative to the corresponding builder.

In one embodiment of the present invention, MGDA and/or GLDA comprised in the detergent formulation may contain in the range of from 0.1 to 10 wt.%, relative to the respective builder, of one or more optically inactive impurities, wherein at least one impurity is selected from iminodiacetic acid, formic acid, glycolic acid, propionic acid, acetic acid and the respective alkali metal or mono-, di-or triammonium salts thereof.

Further examples of detergency builders are polymers having complexing groups, e.g. in which 20 to 90 mol% of the N atoms carry at least one CH₂COO^-Polyethyleneimine of the group, and the corresponding alkali metal salts of the above-mentioned sequestering agents, especially the sodium salts thereof.

Other examples of suitable polymers are polyalkyleneimines, such as polyethyleneimine and polypropyleneimine. The polyalkyleneimines can be used as such or as polyalkoxylated derivatives, such as ethoxylation or propoxylation. The polyalkyleneimine comprises at least 3 alkyleneimine units per molecule.

In one embodiment of the invention, the alkylenimine unit is C₂-C₁₀Alkylene diamine units, e.g. 1, 2-propanediamine, preferably alpha, omega-C₂-C₁₀Alkylene diamines, such as 1, 2-ethylenediamine, 1, 3-propylenediamine, 1, 4-butylenediamine, 1, 5-pentylenediamine, 1, 6-hexylenediamine (also known as 1, 6-hexylenediamine), 1, 8-diamine or 1, 10-decylenediamine, and even more preferably 1, 2-ethylenediamine, 1, 3-propylenediamine, 1, 4-butylenediamine and 1, 6-hexylenediamine.

In another embodiment of the invention, the polyalkyleneimine is selected from polyalkyleneimine units, preferably polyethyleneimine or polypropyleneimine units.

The term "polyethyleneimine" in the context of the present invention relates not only to polyethyleneimine homopolymers, but also to structural units linked to other alkylenediamines, such as NH-CH ₂-CH₂-CH₂-NH structural element, NH-CH₂-CH(CH₃) -NH structural element, NH- (CH)₂)₄-NH structural element, NH- (CH)₂)₆-NH structural element or (NH- (CH)₂)₈The structural units-NH together comprising NH-CH₂-CH₂Polyalkyleneimines of the structural unit-NH-CH₂-CH₂the-NH structural units are predominant in terms of molar proportion. Preferred polyethyleneimines comprise NH-CH₂-CH₂-NH structural units which are predominant in terms of molar fraction, for example 60 mol% or more, for example at least 70 mol%, relative to all alkyleneimine structural units. In a particular embodiment, the term polyethyleneimine relates to polyethyleneimine units having only one or no other than NH-CH per polyethyleneimine unit₂-CH₂-those polyalkyleneimines of alkyleneimine building blocks of NH.

The term "polypropyleneimine" in the context of the present invention relates not only to polypropyleneimine homopolymers, but also to structural units which are linked to other alkylenediamines, for example NH-CH₂-CH₂-CH₂-NH structural element, NH-CH₂-CH₂-NH structural element, NH- (CH)₂)₄-NH structural element, NH- (CH)₂)₆-NH structural element or (NH- (CH)₂)₈The structural units-NH together comprising NH-CH₂-CH(CH₃) Polyalkyleneimines of the structural unit-NH-CH₂-CH(CH₃) the-NH structural units are predominant in terms of molar proportion. Preferred polypropyleneimines comprise NH-CH ₂-CH(CH₃) -NH structural units which are predominant in terms of molar fraction, for example 60 mol% or more, for example at least 70 mol%, relative to all alkyleneimine structural units. In particular embodiments, the term polypropyleneimine relates to units of polypropyleneimine with only one or with no other than NH-CH₂-CH(CH₃) Alkylidene imine structure of-NHThose polyalkyleneimines of the unit.

The branch may be an alkyleneamino group, such as but not limited to-CH₂-CH₂-NH₂Group or (CH)₂)₃-NH₂A group. The longer chain branch may be, for example, - (CH)₂)₃-N(CH₂CH₂CH₂NH₂)₂Or- (CH)₂)₂-N(CH₂CH₂NH₂)₂A group. Highly branched polyethyleneimines are, for example, polyethyleneimines dendrimers or related molecules having a degree of branching in the range from 0.25 to 0.95, preferably from 0.30 to 0.80, particularly preferably at least 0.5. The degree of branching can be determined, for example, by¹³C-NMR or¹⁵N-NMR spectroscopy is preferably carried out in D₂Determined in O and defined as follows:

DB＝D+T/D+T+L

wherein D (dendritic) corresponds to the proportion of tertiary amino groups, L (linear) corresponds to the proportion of secondary amino groups and T (terminal) corresponds to the proportion of primary amino groups.

In the context of the present invention, branched polyethyleneimine units are polyethyleneimine units having a DB in the range from 0.25 to 0.95, particularly preferably from 0.30 to 0.90%, very particularly preferably at least 0.5. Preferred polyethyleneimine units are those with little or no branching, and are therefore predominantly linear or linear polyethyleneimine units.

In the context of the present invention, CH₃The groups are not considered to be branched.

In one embodiment of the invention, the polyalkyleneimines may have a primary amine number in the range of from 1 to 1000mg KOH/g, preferably from 10 to 500mg KOH/g, most preferably from 50 to 300mg KOH/g. Primary amine values can be determined according to ASTM D2074-07.

In one embodiment of the invention, the polyalkyleneimines may have a secondary amine number in the range of from 10 to 1000mg KOH/g, preferably from 50 to 500mg KOH/g, most preferably from 50 to 500mg KOH/g. Secondary amine values can be determined according to ASTM D2074-07.

In one embodiment of the invention, the polyalkyleneimines may have a tertiary amine value in the range of from 1 to 300mg KOH/g, preferably from 5 to 200mg KOH/g, most preferably from 10 to 100mg KOH/g. Tertiary amine number can be determined according to ASTM D2074-07.

In one embodiment of the invention, the molar fraction of tertiary N atoms is determined by¹⁵And (3) N-NMR spectroscopy. In the tertiary amine number and according to¹³When the results of C-NMR spectroscopy are not uniform, the results are preferably obtained by¹³Results obtained by C-NMR spectroscopy.

In one embodiment of the invention, the average molecular weight M of the polyalkyleneimines_wIn the range of 250-100,000g/mol, preferably 250-50,000g/mol, more preferably 800-25,000 g/mol. Average molecular weight M of polyalkyleneimine _wIt can be determined by Gel Permeation Chromatography (GPC) of the intermediate corresponding polyalkyleneimines, with 1.5% by weight aqueous formic acid as eluent and crosslinked polyhydroxyethylmethacrylate as stationary phase.

The polyalkyleneimines can be free or alkoxylated, the alkoxylation being selected from the group consisting of ethoxylation, propoxylation, butoxylation, and combinations of at least two of the foregoing. Preference is given to ethylene oxide, 1, 2-propylene oxide and mixtures of ethylene oxide and 1, 2-propylene oxide. If mixtures of at least two alkylene oxides are used, they can be reacted stepwise or simultaneously.

In one embodiment of the invention, the alkoxylated polyalkyleneimines carry at least 6 nitrogen atoms per unit.

In one embodiment of the invention, the polyalkyleneimines are alkoxylated with 2 to 50 moles of alkylene oxide per NH group, preferably 5 to 30 moles of alkylene oxide per NH group, even more preferably 5 to 25 moles of ethylene oxide or 1, 2-propylene oxide or a combination thereof per NH group. In the context of the present invention, NH₂The units are counted as two NH groups. Preferably all-or almost all-NH groups are alkoxylated and no detectable amount of NH groups remains.

Depending on the preparation of such alkoxylated polyalkyleneimines, the molecular weight distribution can be narrow or broad. For example, the polydispersity Q ═ M _w/M_nIn the range 1 to 3, preferably at least 2, or it may be greater than 3 and up to 20, for example 3.5 to 15, even more preferably in the range 4 to 5.5.

In one embodiment of the invention, the polydispersity Q of the alkoxylated polyalkyleneimines is in the range of from 2 to 10.

In one embodiment of the invention, the alkoxylated polyalkyleneimines are selected from the group consisting of polyethoxylated polyethyleneimines, ethoxylated polypropyleneimines, ethoxylated alpha, omega-hexanediamines, ethoxylated and propoxylated polyethyleneimines, ethoxylated and propoxylated polypropyleneimines, and ethoxylated and polypropoxylated alpha, omega-hexanediamines.

In one embodiment of the invention, the average molecular weight M of the alkoxylated polyethyleneimine_n(number average) in the range of 2,500-1,500,000g/mol, preferably at most 500,000g/mol, as determined by GPC.

In one embodiment of the invention, the average alkoxylated polyalkyleneimines are selected from the group consisting of ethoxylated alpha, omega-hexamethylenediamine and ethoxylated and polypropoxylated alpha, omega-hexamethylenediamine, each having an average molecular weight M_n(number average) is in the range of 800-500,000g/mol, preferably 1,000-30,000 g/mol.

The detergent formulations of the invention may comprise one or more complexing agents other than EDTA, DTPA, MGDA and GLDA, for example citrates, phosphonic acid derivatives, for example disodium salts of hydroxyethane-1, 1-diphosphonic acid ("HEDP"), for example trisodium citrate, and phosphates such as STPP (sodium tripolyphosphate).

In one embodiment, the detergent formulation of the invention comprises a builder system comprising:

ethylenediaminetetraacetic acid (EDTA) and/or diethylenetriaminepentaacetic acid (DTPA) and/or methylglycinediacetic acid (MGDA) and/or glutamic diacetic acid (GLDA) as disclosed above, in an amount in the range of from 0.1 to 25.0 wt%, from 1.0 to 15.0 wt% or from 3.0 to 10.0 wt%, all relative to the total weight of the detergent formulation;

optionally, citric acid in an amount ranging from 0.1 to 10.0 wt.%, from 0.5 to 8.0 wt.%, from 1.0 to 5.0 wt.% or from 2.0 to 4 wt.%, all relative to the total weight of the detergent formulation; the citric acid may be provided as a mixture with formate, for example sodium citrate sodium formate 9: 1;

optionally, at least one phosphonate, preferably selected from derivatives of polyphosphonic acids such as diphosphonic acids, e.g. the sodium salt of HEDP, and derivatives of aminopolyphosphonic acids such as aminoalkylenephosphonic acids, e.g. DTPMP, in an amount in the range of 0.1 to 5.0 wt.%, 0.5 to 3.0 wt.% or 1.0 to 2.0 wt.%, all relative to the total weight of the detergent formulation;

optionally, at least one polycarboxylate selected from homopolymers of unsaturated carboxylic acids having identical repeating units, such as polyacrylic acid (PAA), and copolymers having repeating units of at least two different unsaturated carboxylic acids, such as copolymers of acrylic acid and methacrylic acid, copolymers of acrylic acid or methacrylic acid and maleic acid and/or fumaric acid, in an amount in the range from 0.1 to 10% by weight, from 0.25 to 5% by weight or from 0.3 to 2.5% by weight, all relative to the total weight of the detergent formulation;

In one embodiment of the present invention, the formulations of the present invention are free of phosphates and polyphosphates, wherein hydrogen phosphates are included, e.g. free of trisodium phosphate, pentasodium tripolyphosphate, and hexasodium metaphosphate. In the context of phosphates and polyphosphates, "free" is understood in the context of the present invention to mean that the content of phosphates and polyphosphates is in total in the range from 10ppm to 0.2% by weight, determined gravimetrically and relative to the total weight of the detergent formulation.

The liquid detergent formulations of the present invention may comprise one or more corrosion inhibitors. Non-limiting examples of suitable corrosion inhibitors include sodium silicate, triazoles such as benzotriazoles, dibenzotriazoles, aminotriazoles, alkylaminotriazoles, phenol derivatives such as hydroquinone, catechol, hydroxyhydroquinone, gallic acid, phloroglucinol and 1,2, 3-benzenetrisol, other polyethyleneimines and salts of bismuth or zinc. The corrosion inhibitors may be formulated into the liquid detergent formulations of the present invention in an amount of 0.1 to 1.5 wt.%, relative to the total weight of the liquid detergent formulation.

The liquid detergent formulations of the invention may comprise at least one graft copolymer consisting of:

(a) At least one grafting base selected from the group consisting of non-ionic mono-, di-, oligo-and polysaccharides, and side chains obtained by grafting:

(b) at least one ethylenically unsaturated mono-or dicarboxylic acid, and

(c) at least one compound of formula (XI):

wherein the variables are defined as follows:

R¹selected from the group consisting of methyl and hydrogen,

A¹is selected from C₂-C₄An alkylene group or a substituted alkylene group,

R²are the same or different and are selected from C₁-C₄An alkyl group, a carboxyl group,

X^-selected from halide ions, mono-C₁-C₄Alkyl sulfates and sulfates.

The liquid detergent formulations of the present invention may comprise one or more buffering agents such as monoethanolamine and N, N-triethanolamine.

The foaming characteristics of the liquid detergent formulations of the present invention can be varied to meet various objectives. Hand dishwashing detergents generally require a stable foam. Automatic dishwashing detergents generally require low sudsing. Laundry detergents can range from high sudsing to mild or moderate to low sudsing. Low sudsing laundry detergents are generally recommended for use in front-loading drum washer and washer dryer combinations. The use of foam stabilizers or suds suppressors as detergent components in detergent formulations suitable for specific applications is well known to those skilled in the art. Examples of foam stabilizers include, but are not limited to, alkanolamides and alkyl amine oxides. Examples of suds suppressors include, but are not limited to, alkyl phosphates, silicones, and soaps.

The liquid detergent formulations of the present invention may comprise one or more perfumes such as benzyl salicylate, as well as

Commercially available 2- (4-tert-butylphenyl) -2-methylpropionaldehyde and hexylCinnamic aldehyde.

The liquid detergent formulations of the present invention may comprise one or more dyes such as acid blue 9, acid yellow 3, acid yellow 23, acid yellow 73, pigment yellow 101, acid green 1, solvent green 7 and acid green 25.

The liquid detergent formulation may comprise at least one compound selected from the group consisting of organic solvents, preservatives, viscosity modifiers and hydrotropes.

In one embodiment of the present invention, the liquid detergent formulation comprises an amount of organic solvent of from 0.5 to 25 wt.%, relative to the total weight of the liquid detergent formulation. Especially when the liquid detergent formulation of the present invention is provided in a pouch or the like, the organic solvent may be contained in an amount of 8 to 25% by weight relative to the total weight of the liquid detergent formulation. The organic solvents are those disclosed above.

The liquid detergent formulations of the present invention may comprise one or more preservatives selected from those disclosed above in an amount effective to avoid microbial contamination of the liquid detergent formulation.

In one embodiment of the present invention, the liquid detergent formulation comprises one or more viscosity modifiers. Non-limiting examples of suitable viscosity modifiers include agar, carrageenan, tragacanth, acacia, xanthan gum, alginates, pectin, hydroxyethyl cellulose, hydroxypropyl cellulose, starch, gelatin, locust bean gum, crosslinked poly (meth) acrylates, for example polyacrylic acid crosslinked with di (meth) acrylamide, furthermore silicic acid, clays such as but not limited to montmorillonite, zeolites, dextrins and casein. The viscosity modifier can be included in an amount effective to provide the desired viscosity.

In one embodiment of the invention, the liquid detergent formulation comprises one or more hydrotropes, which may be organic solvents such as ethanol, isopropanol, ethylene glycol, 1, 2-propanediol, and other organic solvents that are water miscible under normal conditions, without limitation. Other examples of suitable hydrotropes are the sodium salts of toluene sulfonic acid, xylene sulfonic acid and cumene sulfonic acid. Hydrotropes may be included in an amount that promotes or enables the dissolution of compounds with limited water solubility.

In one embodiment of the present invention, the formulations according to the invention are free of heavy metal compounds which are not used as bleach catalysts, in particular free of iron compounds. In the context of the present invention, "free" is understood to mean that the content of heavy metal compounds which are not used as bleach catalysts, determined by the leaching method, is in total in the range from 0 to 100ppm, preferably from 1 to 30 ppm. In the context of the present invention, "heavy metals" are all compounds having a specific gravity of at least 6g/cm³Except for zinc and bismuth. Heavy metals are in particular noble metals, and also iron, copper, lead, tin, nickel, cadmium and chromium.

When the liquid detergent formulation of the invention is provided in a divided pouch or the like, the compartment comprising the liquid enzyme preparation of the invention is provided separately from the compartment comprising a bleaching agent, such as an inorganic peroxide or a chlorine-containing bleaching agent, such as sodium hypochlorite. In one embodiment, the compartment comprising the liquid enzyme formulation further comprises at least one complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein MGDA and GLDA are as disclosed above.

In one embodiment, the liquid detergent formulation of the invention is free of bleach, e.g. free of inorganic peroxy compounds or chlorine bleach such as sodium hypochlorite, which means that the liquid detergent formulation of the invention comprises a total of 0.01 wt% or less of inorganic peroxy compounds and chlorine bleach, in each case relative to the total weight of the liquid detergent formulation.

When the solvent contained in the liquid detergent formulation is essentially water, the detergent formulation may be referred to herein as aqueous. In one embodiment, water is the only solvent. In other embodiments, a mixture of water and one or more water-miscible solvents is used. The term water-miscible solvent relates to an organic solvent that is miscible with water at ambient temperature without phase separation. Examples are ethylene glycol, 1, 2-propanediol, isopropanol and diethylene glycol. Preferably at least 50% by volume of water relative to the total solvent contained in the aqueous detergent formulation.

By "detergent formulation" or "cleaning formulation" is meant herein a formulation designated for cleaning soiled materials. Cleaning may refer to laundry or hard surface cleaning. The soiled material comprises a textile and/or a hard surface according to the invention.

The term "laundry" relates to both domestic and industrial laundry and refers to a process for treating textiles with a solution comprising the detergent formulation of the present invention. The laundry process may be carried out by using a process unit such as a domestic or industrial washing machine. Alternatively, the laundry method may be performed manually.

The term "textile" refers to any textile material, including yarns (threads made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, nonwovens, natural materials, synthetic materials, and fabrics made of these materials (textiles made by knitting, or bonding fibers) such as apparel (any article of clothing made of textiles), cloth, and other articles.

The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibres are of vegetable (such as flax, jute and cotton) or animal origin, including proteins such as collagen, keratin and fibroin (e.g. silk, sheep wool, goat wool, mohair, cashmere). Examples of fibres of synthetic origin are polyurethane fibres such as

Or

Polyester fibers, polyolefins such as elastic polyolefins, or polyamide fibers such as nylon. The fibers may be part of a single fiber or a textile such as a knit, woven, or nonwoven.

The term "hard surface cleaning" is defined herein as cleaning of hard surfaces, wherein hard surfaces may include any hard surface in domestic use, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces including cutlery or dishware. Thus, the term "hard surface cleaning" may refer to "dishwashing", the latter involving all forms of dishwashing, such as hand washing or Automatic Dishwashing (ADW). Dishwashing includes, but is not limited to, cleaning all forms of crockery such as dishes, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and utensils and ceramic ware, plastics such as melamine, metals, porcelain, glass and acrylic.

In one aspect, the present invention relates to the provision of a liquid detergent formulation comprising at least an enzyme preparation of the invention and at least one detergent component.

In one embodiment, the present invention provides a liquid detergent formulation comprising at least components (a) and (b) as disclosed above and at least one detergent component, wherein component (b) comprises: at least one amylase selected from the group of alpha-amylases as disclosed above (EC 3.2.1.1), preferably selected from the group consisting of:

And optionally one or more other enzymes selected from the group consisting of proteases, lipases, cellulases and mannanases-all as disclosed above.

In an embodiment of the above embodiment, the liquid detergent formulation has an increased storage stability when compared to a liquid detergent formulation without component (a). Increased storage stability may in this connection mean that the detergent formulation does not have a significant loss of wash performance after storage at 37 ℃ for 1, 2, 4, 6 or 8 weeks on at least one enzyme-sensitive stain type, preferably on at least amylase-sensitive stains.

The wash performance for a given enzyme-sensitive stain type refers to the action of the corresponding enzyme on the enzyme-sensitive portion of the particular stain. Different enzymes are capable of breaking down different types of soils. For example, proteases act on protein materials and thereby degrade proteins into smaller peptides. Amylase-sensitive stains are typically starch-based stains, in which carbohydrates can be degraded by amylase into oligosaccharides or monosaccharides. Lipase-sensitive soils typically comprise fatty compounds. Mannanase-sensitive stains typically comprise mannan. Cellulases can be indirectly cleaned by hydrolyzing certain glycosidic bonds in the cotton fiber. In this way, the particulate dirt adhering to the microfibrils is removed.

No significant wash performance loss after storage may mean that the detergent:

i. at least 90% wash performance after 4 weeks storage at 37 ℃ when compared to the wash performance of the same detergent prior to storage; and/or

Having a wash performance of at least 85% after 6 weeks storage at 37 ℃ when compared to the wash performance of the same detergent prior to storage; and/or

Has a wash performance of at least 80% after 8 weeks of storage at 37 ℃ when compared to the wash performance of the same detergent prior to storage.

In one embodiment, the liquid detergent formulation comprising at least components (a) and (b) and at least one detergent component has an increased storage stability when compared to a liquid detergent formulation without component (a), wherein component (b) comprises at least one amylase preferably selected from the group of alpha-amylases (EC 3.2.1.1) as disclosed above, preferably selected from the group consisting of:

In one embodiment, increased storage stability refers to an increase in wash performance of the liquid detergent formulation after 4-8 weeks of storage at 37 ℃ of at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% when compared to the liquid detergent formulation without component (a) stored at the same temperature for the same time. Increased storage stability may refer to an increase in wash performance of the liquid detergent formulation after 8 weeks of storage at 37 ℃ of at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% when compared to the same time of storage of the liquid detergent formulation without component (a) at the same temperature.

In one embodiment, a liquid detergent formulation comprising at least components (a) and (b) and at least one detergent component has an increased storage stability when compared to a liquid detergent formulation without component (a), wherein component (b) comprises, in addition to at least one alpha amylase as disclosed above, at least one protease as disclosed above, preferably selected from serine endopeptidases (EC 3.4.21), more preferably selected from subtilisin type proteases (EC 3.4.21.62). The protease may be selected from the group consisting of the protease according to SEQ ID No. 22 as described in EP 1921147 or a proteolytically active variant thereof as disclosed above and from subtilisin 309 as disclosed in WO 89/06279 table I a) or a proteolytically active variant thereof as disclosed above.

In one embodiment, the liquid detergent formulation comprising at least components (a) and (b) and at least one detergent component has an increased storage stability when compared to a liquid detergent formulation without component (a), wherein component (b) comprises, in addition to at least one alpha amylase as disclosed above, at least one lipase as disclosed above, preferably selected from the group of fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from thermomyces lanuginosus lipase. In one embodiment, the Thermomyces lanuginosus lipase is selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity.

One aspect of the present invention relates to the use of component (a) for stabilizing component (b) in a liquid detergent formulation preferably comprising at least one complexing agent, preferably in an amount of EDTA and/or DTPA of at most 3 wt%, preferably at most 2.5% and/or in an amount of MGDA and/or GLDA in the range of from 10 to 30 wt%, preferably from 15 to 25%, all relative to the total weight of the liquid detergent formulation, wherein component (b) comprises:

at least one amylase as disclosed above, preferably selected from the group of alpha-amylases as disclosed above (EC 3.2.1.1), more preferably selected from the group consisting of:

and optionally at least one other enzyme, preferably selected from the group consisting of proteases, lipases, cellulases and mannanases-all as disclosed above.

In one embodiment, the at least one protease is selected from serine endopeptidases (EC 3.4.21), more preferably from subtilisin-type proteases (EC 3.4.21.62). The protease may be selected from subtilisin 147 and/or 309 as disclosed in WO 89/06279 or a proteolytically active variant thereof, subtilisin from Bacillus lentus as disclosed in WO 91/02792 or a proteolytically active variant thereof and subtilisin according to SEQ ID NO:22 as described in EP 1921147 or a proteolytically active variant thereof.

In one embodiment, the at least one lipase is selected from fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from thermomyces lanuginosus lipase. In one embodiment, the Thermomyces lanuginosus lipase is selected from the group consisting of the triacylglycerol lipase of amino acids 1 to 269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity.

One aspect of the present invention relates to a method of stabilizing component (b) in a liquid detergent formulation, preferably comprising: EDTA and/or DTPA in an amount of at most 3 wt.%, preferably at most 2.5%, and/or MGDA and/or GLDA in an amount in the range of from 10 to 30 wt.%, preferably from 15 to 25%, all relative to the total weight of the liquid detergent formulation,

wherein component (b) comprises:

By stabilized component (b) is meant in this connection that the wash performance of a liquid detergent formulation comprising component (b) after storage at 37 ℃ for 4 to 8 weeks is increased by at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% against at least one enzyme-sensitive soil, preferably at least one amylase-sensitive soil, when compared to a liquid detergent formulation without component (a) stored at the same temperature for the same time. Stabilizing component (b) may mean that the wash performance of a liquid detergent formulation comprising component (b) after 8 weeks storage at 37 ℃ is increased by at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% when compared to a liquid detergent formulation without component (a) stored at the same temperature for the same time.

One aspect of the present invention relates to the use of component (a) in a liquid detergent formulation to reduce the loss of amylolytic activity of component (b) during storage, preferably during 21, 28 and/or 42 days of storage at 37 ℃, the formulation preferably comprising:

EDTA and/or DTPA in an amount of at most 3 wt.%, preferably at most 2.5%, and/or MGDA and/or GLDA in an amount in the range of from 10 to 30 wt.%, preferably from 15 to 25%, all relative to the total weight of the liquid detergent formulation,

wherein component (b) comprises:

One aspect of the present invention relates to a method of reducing the loss of amylolytic activity of component (b) during storage, preferably during 21, 28 and/or 42 days of storage at 37 ℃, in a liquid detergent formulation, preferably comprising:

wherein component (b) comprises:

The present invention relates in one aspect to a method for increasing the storage stability of a liquid detergent formulation by adding at least one compound of formula (I) to the detergent formulation, which formulation comprises at least one amylase as disclosed above and optionally comprises:

wherein the variables of formula (I) are as follows:

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups ₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H.

In one embodiment, the storage stability of the liquid detergent formulation during 21, 28 and/or 42 days of storage at 37 ℃ is increased when compared to a liquid detergent formulation without the compound of formula (I) stored under the same conditions. Increased storage stability in the present invention may mean an increase in amylolytic stability in the presence of component (a) of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% when compared to amylolytic activity in the absence of component (a).

Other uses

The present invention relates to a method of removing enzyme sensitive stains comprising the step of contacting the stains with a detergent formulation of the invention comprising components (a) and (b) and one or more detergent components, all as disclosed above. In one embodiment, the decontamination method comprises the step of being performed by an automated device such as a washing machine or an automatic dishwasher.

In one embodiment, the detergent formulation comprises the enzyme preparation of the invention.

One aspect of the invention relates to the removal of starch-containing soils. In one embodiment, the removal of the dirt containing starch can be carried out at a cleaning temperature of 40 ℃ or less, at a cleaning temperature of 30 ℃ or less, at a cleaning temperature of 25 ℃ or less, or at a cleaning temperature of 20 ℃ or less.

In one embodiment, the present invention relates to a method for removing starch-containing soils at a cleaning temperature of 30 ℃ or less, wherein the method comprises the step of contacting the soil with a detergent formulation of the invention comprising components (a) and (b) and one or more detergent components. Components (a) and (b) are those disclosed above. In one embodiment, component (b) comprises at least one amylase as disclosed above, preferably selected from the group consisting of alpha-amylases as disclosed above (EC 3.2.1.1), more preferably selected from the group consisting of:

Examples

The invention is further illustrated by working examples.

General description: percentages are by weight unless otherwise specifically indicated.

I.Test compounds

A) A compound of formula (I) — (component (a)):

a.1 triethyl citrate-purchased from Sigma Aldrich

A.2 tripropylcitrate-from Sigma Aldrich

A.3 Tributyl citrate-from Sigma Aldrich

A.4 Acetyltributyl citrate-from Sigma Aldrich

A.5 acetyl triethyl citrate-purchased from Sigma Aldrich

A.6 citric acid monoethyl ester-purchased from Sigma Aldrich A.7 diethyl citrate

Synthesis as described in Journal of Chemical & Engineering Data 2018, DOI:10.1021/acs.jced.7b01060, C.Berdggo, A.Suaza, M.Santaella, O.Sanchez

A.8 Tribenzyl citrate

Synthesized as described in WO2007/14471 a1, 2007; position in the patent: page 19, columns 27-28;

a.9 Trisalicyl citrate

B) comparative compound (c):

b.1: citric acid-purchased from Sigma Aldrich

B.2: trisodium citrate-purchased from Sigma Aldrich

B.3: oxalic acid diethyl ester-purchased from Sigma Aldrich

B.4: glycerol triacetate (triacetin) -available from Sigma Aldrich

II.Stability of Amylase and protease

The storage stability of the amylases was evaluated at 37 ℃.

Base test formulations were prepared by mixing the components to prepare base formulations I-V according to table 1.

The amylase used was: amy1 ═ Stainzyme, Amy2 ═ Amplify, Amy3 ═ Stainzyme Plus L (12L)

The protease used was: (S) Savinase Ultra 16.0L (CAS No. 9014-01-1, EC No. 232-.

If applicable, the respective component (a) or the comparative compound is added to the respective base formulation in the amounts indicated in table 1.

The amylase (component (b)) was added to the respective base formulation in the amounts shown in table 1. The amylase amounts provided in table 1 relate to active protein.

The protease enzyme (component (b)) was added to the corresponding base formulation in the amounts shown in table 1. The protease amounts provided in table 1 relate to the active protein.

Water was added to achieve equilibrium to 100.

Table 1: liquid formulations

(Comp.1)：Trilon M fl(Max liqu)

(Comp.2): citric acid

(Comp.3): GLDA 50% solution

(Comp.4): PAA, polyacrylic acid Mw 5.000g/mol

(Comp.5): glycerol (G) or propylene glycol (P)

(Comp.6)：Dehypon WET

(Comp.7)：Na₄HEDP

(Comp.8) thickener (Rheotare XGN)

For comparative experiments without the compounds of the invention, they were replaced with the same amount of glycerol.

The amylase activity at certain time points shown in Table 2 was quantitatively measured by the release of the chromophore p-nitrophenol (pNP) from the substrate (ethylene protected pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and alpha-glucosidase (Roche Applied Science 11626329103) present in excess compared to the alpha-amylase, processing these smaller products until pNP is released; the release of pNP measured via an increase in absorbance at 405nm is directly proportional to the alpha-amylase activity of the sample. Amylase standards: termamyl 120L (Sigma 3403).

Table 2 shows the amylase activity measured in the liquid formulation after 1-30 days of storage at 37 ℃. The amylolytic activity values provided are calculated with reference to the values determined at time 0 in the reference formulation.

The formulations are named as follows: the Roman numerals preceding the period characterize the base formulation, and the Arabic numerals characterize the compound type (A. # compounds of the invention (component (a)); B. # comparative compounds).

Table 2: amylase Activity during storage time at 37 ℃

Protease activity at certain time points shown in Table 3 was determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, abbreviated AAPF) as a substrate. pNA is separated from the substrate molecule by proteolytic cleavage, resulting in the release of yellow free pNA, which can be measured by OD₄₀₅And then measured. The measurement was carried out at 20 ℃.

Table 3 shows the protease activity measured in the liquid formulation after 1-30 days of storage at 37 ℃. The proteolytic activity values provided in table 3 are calculated with reference to the values determined at time 0 in the reference formulation.

The formulations are named as follows: the Roman numerals preceding the period characterize the base formulation, and the Arabic numerals characterize the salt type (A. # salts of the invention (component (a)); B. # comparative compound). Zero ("0"): no salt, but diethylene glycol.

Table 3: protease activity during storage time at 37 ℃

III.Stability in laundry formulations

The storage stability of the amylases was evaluated at 37 ℃.

Base test formulations were prepared by mixing the components to prepare base formulations VI-IX according to table 4.

Table 4: liquid laundry formulations

(Comp.1)：n-C₁₈Alkyl- (OCH)₂CH₂)₂₅-OH

(Comp.2): tallow oil soap C₁₄-C₁₈Sodium salt of carboxylic acid

(Comp.3)：C₁₀-C₁₂Sodium alkyl benzene sulfonate

(Comp.4): sodium laureth sulfate-n-C₁₂H₂₅-O-(CH₂CH₂O)₃-SO₃Na

(Comp.5): complexing agent EDTA or DTPA

(Comp.6) sodium citrate sodium formate mixture 9:1

For comparative experiments without the compounds of the invention, they were replaced with the same amount of water.

The amylase activity at certain time points shown in Table 5 was quantitatively measured by the release of the chromophore p-nitrophenol (pNP) from the substrate (ethylene protected pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and alpha-glucosidase (Roche Applied Science 11626329103) present in excess compared to the alpha-amylase, processing these smaller products until pNP is released; the release of pNP measured via an increase in absorbance at 405nm is directly proportional to the alpha-amylase activity of the sample. Amylase standards: termamyl 120L (Sigma 3403).

Table 5 shows the amylase activity measured in the liquid formulation after 1-28 days of storage at 37 ℃. The amylolytic activity values provided are calculated with reference to the values determined at time 0 in the reference formulation.

Table 5: amylase Activity during storage time at 37 ℃

The wash performance of the formulations according to table 4 in cleaning amylase-sensitive stains can be carried out on test fabrics of the type suitable for use. Pre-stained test fabrics are available from wfk test fabrics GmbH, Krefeld; EMPA is Swiss Federal Institute of Materials Testing; or CFT for Test Material b.v.

The test may be performed as follows: a multiple soil monitor comprising, for example, 8 standardized pieces of soiled fabric, each 2.5 x 2.5cm in size and sewn on both sides to a polyester carrier, was washed in a jar soil test machine with 2.5g cotton fabric and 5g/L liquid test laundry detergent, table 4.

The conditions may be selected as follows: the device comprises the following steps: a jar detergency tester from SDL Atlas, Rock Hill, USA; washing liquid: 250ml, washing time: 60 minutes, washing temperature: at 30 ℃. Water hardness: 2.5 mmol/L; ca, Mg, HCO ₃4:1: 8; fabric/wash ratio 1: 12; the multiple soil monitor was rinsed in water after the wash cycle and then dried at ambient temperature for a period of 14 hours.

The overall level of cleanliness can be assessed using color measurements. The reflectance values of the soiling on the monitor were measured at 460nm using a spherical reflectance spectrometer (model SF 500 from Datacolor, USA, wavelength range 360-. In this case, the brightness L, the a values on the red-green color axis and the b values on the yellow-blue color axis were measured before and after washing by means of CIE-Lab color space classification and averaged over 8 soiling of the monitor. The color value change (Δ E) value may be automatically defined and calculated by the evaluation color tool based on the following equation:

[ lightness, color a on the red-green axis, color b on the blue-yellow axis ]

Δ E is a measure of the cleaning effect achieved. All measurements can be repeated 6 times to get an average. Note that higher Δ Ε values indicate better cleaning. The difference of 1 unit can be detected by the skilled person. Non-experts can easily detect 2 units.

The jar detergency tester tests were performed with the freshly prepared formulations according to table 4 and/or the same formulations stored at 37 ℃ for a defined time, such as 3 days, about 7 days, about 14 days, about 21 days, about 28 days, or >28 days. As an approximation, 1 week (7 days) at 37 ℃ equals 31/2 weeks at 20 ℃.

Claims

1. An enzyme preparation comprising:

component (a): at least one compound of the general formula (I):

wherein the variables in formula (I) are defined as follows:

R¹selected from H, acetyl and propionyl;

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H;

a component (b): at least one enzyme selected from hydrolases (EC 3), preferably at least one enzyme selected from amylases, more preferably at least one enzyme selected from alpha-amylases (EC 3.2.1.1); and/or at least one enzyme selected from proteases, preferably from proteases of the subtilisin type (EC 3.4.21.62),

and optionally

A component (c): a compound selected from at least one solvent, at least one enzyme stabilizer different from component (a) and at least one compound stabilizing the liquid enzyme formulation itself.

2. The enzyme preparation according to claim 1, wherein the enzyme preparation comprises component (a) in an amount in the range of 0.1-30 wt. -%, relative to the total weight of the enzyme preparation.

3. The enzyme preparation according to claim 1 or 2, characterized in that at least one hydrolase, preferably a hydrolase selected from the group consisting of alpha-amylase and subtilisin-type proteases, comprised in component (b) is stabilized when compared to the enzyme preparation without component (a).

4. A method of preparing a stabilized enzyme preparation, said method comprising the step of mixing at least the following components:

component (a): at least one compound of the general formula (I):

wherein the variables in formula (I) are defined as follows:

R¹selected from H, acetyl and propionyl;

R²、R³、R⁴independently of each other, selected from H, linear C₁-C₈Alkyl and branched C₃-C₈Alkyl, C unsubstituted or substituted by one or more carboxylic acid groups or hydroxy groups₆-C₁₀Aryl and C₆-C₁₀Arylalkyl, wherein the latter alkyl is selected from linear C₁-C₈Alkyl or branched C₃-C₈Alkyl radical, wherein R²、R³And R⁴At least one of which is not H,

a component (b): at least one enzyme selected from hydrolases (EC 3), preferably at least one enzyme selected from amylases, more preferably at least one enzyme selected from alpha-amylases (EC 3.2.1.1); and/or at least one enzyme selected from proteases, preferably from subtilisin-type proteases (EC 3.4.21.62);

and optionally

5. A method of reducing the loss of amylolytic activity of at least one amylase comprised in a liquid enzyme preparation during storage by the step of adding at least one compound of formula (I):

Wherein the variables in formula (I) are defined as follows:

R¹selected from H, acetyl and propionyl;

6. Use of a compound of formula (I) as an additive to a composition comprising at least one hydrolase, preferably a hydrolase selected from the group consisting of alpha-amylases and subtilisin-type proteases:

wherein the variables in formula (I) are defined as follows:

R¹selected from H, acetyl and propionyl;

wherein the compound of formula (I) and the amylase are solids and wherein the amylolytic activity of the amylase is stabilized upon contact of the compound of formula (I) and the amylase with at least one solvent.

7. Use of an enzyme preparation according to any of claims 1 to 3 formulated into a detergent formulation, preferably a liquid detergent formulation, wherein the enzyme preparation according to any of claims 1 to 3 is mixed in one or more steps with one or more detergent components and wherein the detergent formulation preferably comprises at least one complexing agent, preferably selected from EDTA, DTPA, MGDA and GLDA, in an effective amount.

8. Detergent formulation comprising an enzyme preparation according to any of claims 1 to 3 and at least one detergent component, preferably at least one complexing agent, preferably selected from EDTA, DTPA, MGDA and GLDA, in effective amounts.

9. A method of making a detergent formulation comprising the step of mixing at least the following components in effective amounts:

component (a): at least one compound of the general formula (I):

wherein the variables of formula (I) are as follows:

R¹selected from H, acetyl and propionyl;

and at least one detergent component, preferably at least one complexing agent preferably selected from EDTA, DTPA, MGDA and GLDA.

10. A process according to claim 9, comprising the step of mixing the enzyme preparation according to any of claims 1-3 and at least one detergent component, preferably at least one complexing agent, preferably selected from EDTA, DTPA, MGDA and GLDA, in effective amounts.

11. A method for removing amylase-sensitive stains comprising the step of contacting at least one stain with a detergent formulation according to claim 8, wherein component (b) of the detergent formulation comprises at least one alpha-amylase and optionally further comprises at least one subtilisin-type protease.

12. The method according to claim 11, wherein the amylase-sensitive soil is removed from the textile at a cleaning temperature of 30 ℃ or less.

13. A method for increasing the storage stability of a liquid detergent formulation comprising at least one hydrolase and optionally at least one complexing agent, preferably selected from EDTA, DTPA, MGDA and GLDA, in an effective amount by adding at least one compound of formula (I):

wherein the variables of formula (I) are as follows:

R¹selected from H, acetyl and propionyl;

14. The method according to claim 13, wherein the detergent is stored at 37 ℃ for at least 20 days.

15. The method according to claim 13 or 14, wherein the at least one hydrolase (EC 3) is selected from the group consisting of alpha-amylase (EC 3.2.1.1) and subtilisin-type protease (EC 3.4.21.62) and combinations thereof.